ACIDES NUCLEIQUES D'IL-12 MONOCATENAIRES, POLYPEPTIDES D'IL-12 MONOCATENAIRES, ET LEURS UTILISATIONS

SINGLE CHAIN IL-12 NUCLEIC ACIDS, POLYPEPTIDES, AND USES THEREOF

Abrégé français

La présente invention concerne de nouveaux polypeptides d'interleukine-12 monocaténaires. L'invention concerne également des acides nucléiques isolés codant pour les polypeptides d'interleukine-12 monocaténaire, des vecteurs et des cellules les comprenant, ainsi que leurs utilisations, en particulier dans des procédés d'utilisation des polypeptides d'IL-12 monocaténaire, des polynucléotides, des vecteurs et des cellules selon l'invention pour améliorer la fonction du système immunitaire, par exemple comme adjuvants de vaccins et dans le traitement des infections et du cancer.

Abrégé anglais

The present invention relates to novel single chain interleukin-12 polypeptides. T he invention also relates to isolated nucleic acids encoding the single chain interleukin-12 polypeptides, to vectors and cells comprising them, and to their uses, in particular in methods of using single chain IL-12 polypeptides, polynucleotides, vectors and cells of the invention for enhancing immune system function, for example as vaccine adjuvants and in the treatment of infections and cancer.

Revendications

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CLAIMS
The invention claimed is:
1. A single-chain IL-12 polypeptide comprising, from N- to C-terminus:
i. a first IL-12 p40 domain (p40N),
ii. an optional first peptide linker,
iii. an IL-12 p35 domain,
iv. a optional second peptide linker, and
v. a second IL-12 p40 domain (p40C);
wherein the first IL-12 p40 domain (p40N) is an N-terminal fragment of a p40
subunit;
the IL-12 p35 domain is a mature p35 subunit or fragment thereof; and the
second IL-12
p40 domain (p40C) is a C-terminal fragment of a p40 subunit.
2. The single chain IL-12 polypeptide of claim 1, which comprises an N-
terminal signal
peptide domain.
3. The single chain IL-12 polypeptide of claim 1, wherein the polypeptide
does not comprise
a first peptide linker, does not comprise a second peptide linker, or does not
comprise a
first peptide linker and does not comprise a second peptide linker.
4. The single chain IL-12 polypeptide of claim 2, wherein the polypeptide
does not comprise
a first peptide linker, does not comprise a second peptide linker, or does not
comprise a
first peptide linker and does not comprise a second peptide linker.
5. The single chain IL-12 polypeptide of claim 1, wherein said polypeptide
comprises an
amino acid sequence at least 80% identical, at least 85% identical, at least
90% identical,
at least 95% identical, at least 97% identical, at least 98% identical, or at
least 99%
identical to the polypeptide sequence of amino acids 23 to 533 of SEQ ID NO:
10.
6. The single chain IL-12 polypeptide of claim 5, comprising amino acids 23
to 533 of SEQ
ID NO: 10.

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7. The single chain IL-12 polypeptide of claim 2, w herein said polypeptide
comprises an
amino acid sequence at least 80% identical, at least 85% identical, at least
90% identical,
at least 95% identical, at least 97% identical, at least 98% identical, or at
least 99%
identical to the polypeptide sequence of SEQ ID NO: 10.
8. The single chain IL-12 polypeptide of claim 7, comprising the amino acid
sequence of
SEQ ID NO: 10.
9. The single chain IL-12 polypeptide of claim 1, wherein the first and
second peptide
linkers each comprise a number of amino acid residues selected from the group
consisting
of:
a) 0 amino acids;
b) 1 amino acid;
c) 2 amino acids;
d) 3 amino acids;
e) 4 amino acids;
f) 5 amino acids;
g) 6 amino acids;
h) 7 amino acids;
i) 8 amino acids;
j) 9 amino acids; and,
k) 10 amino acids.
10. The single chain IL-12 polypeptide of claim 9, wherein the amino acid
residues in either
the first or second peptide linker, or in both peptide linkers, comprise any
combination of
one or more amino acids selected from the group consisting of:
a) Glycine (Gly);
b) Serine (Ser);
c) Alanine (Ala);
d) Threonine (Thr); and,
e) Proline (Pro).

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11 . The single chain IL-12 polypeptide of claim 1, wherein the first and
second peptide
linkers are selected from Thr-Pro-Ser (SEQ ID NO: 41) and Ser-Gly-Pro-Ala-Pro
(SEQ
ID NO: 42).
12. The single chain IL-12 polypeptide of claim 1, w herein p40N comprises
a polypeptide
sequence at least 80% identical, at least 85% identical, at least 90%
identical, at least 95%
identical, at least 97% identical, at least 98% identical, or at least 99%
identical to amino
acids 18 to 288, amino acids 18 to 298, amino acids 28 to 288, or amino acids
28 to 298
of SEQ ID NO: 2.
13. The single chain IL-12 polypeptide of claim 1, wherein p40N comprises a
fragment of the
polypeptide sequence of SEQ ID NO: 2.
14. The single chain IL-12 polypeptide of claim 13, wherein p40N comprises
a fragment of
the polypeptide sequence of SEQ ID NO: 2, wherein the first residue of said
fragment
begins at a position selected from the group consisting of:
a) amino acid residue 18 of SEQ ID NO: 2;
b) amino acid residue 19 of SEQ ID NO: 2;
c) amino acid residue 20 of SEQ ID NO: 2;
d) amino acid residue 21 of SEQ ID NO: 2;
e) amino acid residue 22 of SEQ ID NO: 2;
f) amino acid residue 23 of SEQ ID NO: 2;
g) amino acid residue 24 of SEQ ID NO: 2;
h) amino acid residue 25 of SEQ ID NO: 2;
i) amino acid residue 26 of SEQ ID NO: 2;
j) amino acid residue 27 of SEQ ID NO: 2; and,
k) amino acid residue 28 of SEQ ID NO: 2,
and, wherein the last residue of said fragment ends at a position selected
from the group
consisting of:
l) amino acid residue 288 of SEQ ID NO: 2;
m) amino acid residue 289 of SEQ ID NO: 2;
n) amino acid residue 290 of SEQ ID NO: 2;

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o) amino acid residue 291 of SEQ ID NO: 2;
p) amino acid residue 292 of SEQ ID NO: 2;
q) amino acid residue 293 of SEQ ID NO: 2;
r) amino acid residue 294 of SEQ ID NO: 2;
s) amino acid residue 295 of SEQ ID NO: 2;
t) amino acid residue 296 of SEQ ID NO: 2;
u) amino acid residue 297 of SEQ ID NO: 2; and,
v) amino acid residue 298 of SEQ ID NO: 2.
15. The single chain IL-12 polypeptide of claim 1, w herein p35 comprises a
polypeptide
sequence at least 80% identical, at least 85% identical, at least 90%
identical, at least 95%
identical, at least 97% identical, at least 98% identical, or at least 99%
identical to amino
acids 52 to 247, amino acids 52 to 253, amino acids 62 to 247, or amino acids
62 to 253
of SEQ ID NO: 4.
16. The single chain IL-12 polypeptide of claim 1, wherein p35 comprises a
fragment of the
polypeptide sequence of SEQ ID NO: 4.
17. The single chain IL-12 polypeptide of claim 16, wherein p35 comprises a
fragment of the
polypeptide sequence of SEQ ID NO:4, wherein the first residue of said
fragment begins
at a position selected from the group consisting of:
a) amino acid residue 52 of SEQ ID NO: 4;
b) amino acid residue 53 of SEQ ID NO: 4;
c) amino acid residue 54 of SEQ ID NO: 4;
d) amino acid residue 55 of SEQ ID NO: 4;
e) amino acid residue 56 of SEQ ID NO: 4;
f) amino acid residue 57 of SEQ ID NO: 4;
g) amino acid residue 58 of SEQ ID NO: 4;
h) amino acid residue 59 of SEQ ID NO: 4;
i) amino acid residue 60 of SEQ ID NO: 4;
j) amino acid residue 61 of SEQ ID NO: 4; and,
k) amino acid residue 62 of SEQ ID NO: 4,

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and, wherein the last residue of said fragment ends at a position selected
from the group
consisting of:
l) amino acid residue 247 of SEQ ID NO: 4;
m) amino acid residue 248 of SEQ ID NO: 4;
n) amino acid residue 249 of SEQ ID NO: 4;
o) amino acid residue 250 of SEQ ID NO: 4;
p) amino acid residue 251 of SEQ ID NO: 4;
q) amino acid residue 252 of SEQ ID NO: 4; and,
r) amino acid residue 253 of SEQ ID NO: 4.
18. The single chain IL-12 polypeptide of claim 1, wherein p40C comprises a
polypeptide
sequence at least 80% identical, at least 85% identical, at least 90%
identical, at least 95%
identical, at least 97% identical, at least 98% identical, or at least 99%
identical to amino
acids 289 to 322, amino acids 289 to 328, amino acids 299 to 322, or amino
acids 299 to
328 of SEQ ID NO: 2.
19. The single chain IL-12 polypeptide of claim 1, wherein p40C comprises a
fragment of the
polypeptide sequence of SEQ ID NO: 2.
20. The single chain IL-12 polypeptide of 19, wherein p40C comprises a
fragment of the
polypeptide sequence of SEQ ID NO: 2, wherein the first residue of said p40C
fragment
begins at a position selected from the group consisting of:
a) amino acid residue 289 of SEQ ID NO: 2;
b) amino acid residue 290 of SEQ ID NO: 2;
c) amino acid residue 291 of SEQ ID NO: 2;
d) amino acid residue 292 of SEQ ID NO: 2;
e) amino acid residue 293 of SEQ ID NO: 2;
f) amino acid residue 294 of SEQ ID NO: 2;
g) amino acid residue 295 of SEQ ID NO: 2;
h) amino acid residue 296 of SEQ ID NO: 2;
i) amino acid residue 297 of SEQ ID NO: 2;
j) amino acid residue 298 of SEQ ID NO: 2; and,

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k) amino acid residue 299 of SEQ ID NO: 2,
and, wherein the last residue of said fragment ends at a position selected
from the group
consisting of:
l) amino acid residue 322 of SEQ ID NO: 2;
m) amino acid residue 323 of SEQ ID NO: 2;
n) amino acid residue 324 of SEQ ID NO: 2;
o) amino acid residue 325 of SEQ ID NO: 2;
p) amino acid residue 326 of SEQ ID NO: 2;
q) amino acid residue 327 of SEQ ID NO: 2; and,
r) amino acid residue 328 of SEQ ID NO: 2.
21. A polynucleotide comprising a nucleic acid sequence encoding the single
chain IL-12
polypeptide of any one of claims 1 to 20.
22. The polynucleotide of claim 21, w herein the polynucleotide comprises a
nucleic acid
sequence at least 80% identical, at least 80% identical, at least 85%
identical, at least 90%
identical, at least 95% identical, at least 97% identical, at least 98%
identical, at least 99%
identical to nucleotides 67 to 1599 of SEQ ID NO: 9.
23. The polynucleotide of claim 22, wherein the polynucleotide comprises
nucleic acids 67 to
1599 of SEQ ID NO: 9.
24. A vector comprising the polynucleotide of any one of claims 21 to 23.
25. The vector of claim 24, wherein the vector further comprises a gene
switch capable of
regulating expression of the single-chain IL-12 polypeptide.
26. The vector of claim 25, wherein the gene switch is an EcR-based gene
switch.
27. The vector of any one of claims 24 to 26, wherein the vector is an
adenovirus, an adeno-
associated virus, a retrovirus, or a lentivirus.

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28. An isolated host cell or a non-human organism transformed or
transfected with the vector
of any one of claims 24 to 27.
29. The isolated host cell of claim 28, wherein the host cell is an immune
cell or a stem cell.
30. The isolated host cell of claim 29, wherein the immune cell is a
dendritic cell, a
macrophage cell, a neutrophil cell, a mast cell, an eosinophil cell, a
basophil cell, a
natural killer cell, or a lymphocyte cell.
31. The isolated host cell of claim 30, wherein the lymphocyte cell is a T
cell.
32. The isolated host cell of claim 29, wherein the stem cell is a
mesenchymal stem cell, an
endometrial stem cell, or an embryonic stem cell.
33. The isolated host cell of claim 32, wherein the endometrial stem cell
is an endometrial
regenerative cell (ERC).
34. A method of treating a patient comprising administering an effective
amount of the single
chain IL-12 polypeptide of any one of claims 1 to 20.
35. A method of treating a patient comprising administering an effective
amount of the
polynucleotide of any one of claims 21 to 23.
36. A method of treating a patient comprising administering an effective
amount of the vector
of any one of claims 24 to 27.
37. A method of treating a patient comprising administering an effective
amount of the host
cell of of any one of claims 28 to 33.

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38. A medicament comprising the polypeptide of any one claims 1 to 20, the
polynucleotide
of any one of claims 21 to 23, the vector of any one of claims 24 to 27, or
the isolated
host cell of any one of claims 28 to 33.
39. The medicament of claim 38, wherein said medicament is administered in
an effective
amount to enhance the immune response in a patient in need thereof.
40. The medicament of claim 38, wherein said medicament is useful for the
treatment of a
condition selected from the group consisting of: c ancer, an infectious
disease, and an
immune system disorder.
41. The medicament of claim 40, wherein the cancer is selected from the
group consisting of:
breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer,
colon cancer,
melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain
carcinoma,
head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-
small cell lung
cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung
carcinoma,
small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular
carcinoma,
bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma,
prostatic
carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma,
myeloma,
multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial
carcinoma,
adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid
carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia,
cervical
hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic
leukemia, acute
myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic
leukemia,
acute granulocytic leukemia, hairy cell leukemia, neuroblastoma,
rhabdomyosarcoma,
Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's
disease, non-
Hodgkin's lymphoma, soft-tissue sarcoma, mesothelioma, osteogenic sarcoma,
primary
macroglobulinemia, and retinoblastoma.

Description

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SINGLE CHAIN IL-12 NUCLEIC ACIDS, POLYPEPTIDES, AND USES
THEREOF
REFERENCE TO SEQUENCE LISTING
[0001] The content of the electronically submitted sequence listing (Name:
SequenceListing.txt; Size: 127,325 bytes; Date of Creation: December 16, 2013)
filed
with this application is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention provides novel nucleic acids encoding single-
chain
interleukin-12 fusion proteins, vectors comprising them, polypeptides encoded
by them,
and for their use in therapeutic applications.
BACKGROUND OF THE INVENTION
[0003] Interleukin-12 (IL-12) is an inflammatory cytokine that is produced
in response to
infection by a variety of cells of the immune system, including phagocytic
cells, B cells
and activated dendritic cells (Colombo and Trinchieri (2002), Cytokine &
Growth Factor
Reviews, 13: 155-168). IL-12 plays an essential role in mediating the
interaction of the
innate and adaptive arms of the immune system, acting on T-cells and natural
killer (NK)
cells, enhancing the proliferation and activity of cytotoxic lymphocytes and
the
production of other inflammatory cytokines, especially interferon-gamma (IFN-
gamma).
[0004] IL-12 has been tested in human clinical trials as an
immunotherapeutic agent for
the treatment of a wide variety of cancers (Atkins et al. (1997), Clin. Cancer
Res., 3: 409-
17; Gollob et al. (2000), Clin. Cancer Res., 6: 1678-92; Hurteau et al.
(2001), Gynecol.
Oncol., 82: 7-10; and Youssouflan, et al. (2013) Surgical Oncology Clinics of
North
America, 22(4): 885-901), including renal, colon, and ovarian cancer, melanoma
and T-
cell lymphoma, and as an adjuvant for cancer vaccines (Lee et al. (2001), J.
Clin. Oncol.
19: 3836-47). However, IL-12 is toxic when administered systemically as a
recombinant
protein. T rinchieri, Adv. Immunol. 1998; 70:83-243. In order to maximize the
anti-
tumoral effect of IL-12 while minimizing its systemic toxicity, IL-12 gene
therapy
approaches have been proposed to allow production of the cytokine at the tumor
site,

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thereby achieving high local levels of IL-12 with low serum concentration.
Qian et al.,
Cell Research (2006) 16: 182-188; US Patent Publication 20130195800.
[0005] IL-12 is a heterodimeric molecule composed of an alpha chain (the
p35 subunit)
and a beta chain (the p40 subunit) covalently linked by a disulfide bridge to
form the
biologically active 70 kD a dimer. S imultaneous expression of the two
subunits is
necessary for the production of the biologically active heterodimer.
Recombinant IL-12
expression has been achieved using bicistronic vectors containing the p40 a nd
p35
subunits separated by an IRES (internal ribosome entry site) sequence to allow
independent expression of both subunits from a single vector. However, the use
of IRES
sequences can impair protein expression. Mizuguchi et al. Mol Ther (2000); 1:
376-382.
Moreover, unequal expression of the p40 and p35 subunits can lead to the
formation of
homodimeric proteins (e.g. p40-p40) which can have inhibitory effects on IL-12
signaling. Gillessen et al. Eur. J. Immunol. 1995 Jan;25(1):200-6.
[0006] As an alternative to bicistronic expression of the IL-12 subunits,
functional single
chain IL-12 fusion proteins have been produced by joining the p40 and p35
subunits with
(Gly4Ser)3 or Gly6Ser linkers. Lieschke et al., (1997), Nature Biotechnology
15, 35-40;
Lode et al., (1998), PNAS 95, 2475 -2480. H owever, longer linker sequences
may
interfere with the ability to construct viral vectors for gene therapy, and
may increase the
likelihood of inducing immunogenic responses (e.g., by generating anti-single
chain IL-
12 antibodies).
[0007] Therefore, there remains a need in the art for improved single
chain IL-12 fusion
proteins and nucleic acids encoding such fusion proteins for use in enhancing
immune
system function, for example as vaccine adjuvants and in the treatment of
infections and
cancer.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention relates to novel single chain IL-12 (scIL-12)
polypeptides
wherein the length of linker sequences, if any, is minimized by inserting IL-
12 p35
polypeptide sequences within an IL-12 p40 polypeptide sequence while retaining
at least
one IL-12 biological activity.
[0009] The present invention relates to scIL-12 polypeptides comprising,
from N- to C-
terminus:

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(i) a first IL-12 p40 domain (p4ON),
(ii) an optional first peptide linker,
(iii) an IL-12 p35 domain,
(iv) a optional second peptide linker, and
(v) a second IL-12 p40 domain (p40C).
[0010] In preferred embodiments, scIL-12 polypeptides of the invention
retain at least
one biological activity of IL-12.
[0011] The invention further relates to scIL-12 polynucleotides encoding
scIL-12
polypeptides as described herein, and to vectors comprising said scIL-12
polynucleotides.
[0012] The invention also relates to variant scIL-12 polypeptides having
80%, 85%, 90%,
or 95% identity to a scIL-12 polypeptide disclosed herein.
[0013] The invention also relates to a cell or a non-human organism
transformed or
transfected with a scIL-12 polynucleotide or vector as described herein.
[0014] The invention also relates to a pharmaceutical or diagnostic
composition
comprising as an active agent a scIL-12 polypeptide, polynucleotide, vector,
or cell as
described herein.
[0015] The invention also relates to methods of using scIL-12
polypeptides,
polynucleotides, vectors and cells of the invention for enhancing immune
system
function, for example as vaccine adjuvants and in the treatment of infections
and cancer.
DESCRIPTION OF THE FIGURES
[0016] Figure 1. S chematic diagrams showing the p40-p35 single chain
configuration
(Fig. 1A), the p35-p40 single chain configuration (Fig. 1B), and a p4ON-p35-
p40C insert
configuration (Fig. 1C). The construction and characterization of these
designs are
discussed in detail in the Examples.
[0017] Figure 2. E xpression levels of human scIL-12 designs as determined
by p70
ELISA (see Example 2).
[0018] Figure 3. scIL-12-stimulated IFN-gamma production, as measured by
ELISA (see
Example 3).
[0019] Figure 4. In vitro dose-dependent expression of interferon-gamma
(i.e., "IFN-
gamma," "IFN-g" or "IFN-y") by NK92 cells exposed to recombinant human or
mouse
IL-12 p40/p35 heterodimeric polypeptides (see Example 4).

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[0020] Figure 5. In vitro dose-dependent expression of interferon-gamma by
NK92 cells
exposed to heterodimeric IL-12 p40/p35 polypeptides and single chain IL-12
polypeptides (including p4ON-p35-p40C single chain IL-12 (SEQ ID NO:10)); (see
Example 5).
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention advantageously provides isolated
polynucleotides encoding
single chain IL-12 (scIL-12) polypeptides. The polynucleotides and
polypeptides of the
present invention are useful in methods of enhancing the immune response of a
host, for
example as vaccine adjuvants, and in the treatment of proliferative disorders
such as
cancer, infectious diseases, and immune system disorders.
[0022] The various aspects of the invention will be set forth in greater
detail in the
following sections, directed to the nucleic acids, polypeptides, vectors,
compositions,
antibodies and methods of use of the invention. This organization into various
sections is
intended to facilitate understanding of the invention, and is in no way
intended to be
limiting thereof.
Definitions
[0023] The following defined terms are used throughout the present
specification, and
should be helpful in understanding the scope and practice of the present
invention.
[0024] In a specific embodiment, the term "about" or "approximately" means
within 20%,
preferably within 10%, more preferably within 5%, and even more preferably
within 1%
of a given value or range.
[0025] The term "substantially free" means that a composition comprising
"A" (where
"A" is a single protein, DNA molecule, vector, recombinant host cell, etc.) is
substantially
free of "B" (where "B" comprises one or more contaminating proteins, DNA
molecules,
vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors
(depending on the category of species to which A and B belong) in the
composition is
"A". Preferably, "A" comprises at least about 90% by weight of the A+B species
in the
composition, most preferably at least about 99% by weight. It is also
preferred that a
composition, which is substantially free of contamination, contain only a
single molecular
weight species having the activity or characteristic of the species of
interest.

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[0026] The term "isolated" for the purposes of the present invention
designates a
biological material (nucleic acid or protein) that has been removed from its
original
environment (the environment in which it is naturally present). F or example,
a
polynucleotide present in the natural state in a plant or an animal is not
isolated, however
the same polynucleotide separated from the adjacent nucleic acids in which it
is naturally
present, is considered "isolated". The term "purified" does not require the
material to be
present in a form exhibiting absolute purity, exclusive of the presence of
other
compounds. It is rather a relative definition.
[0027] A polynucleotide is in the "purified" state after purification of
the starting material
or of the natural material by at least one order of magnitude, preferably 2 or
3 a nd
preferably 4 or 5 orders of magnitude.
[0028] As used herein, the term "substantially pure" describes a
polypeptide or other
material which has been separated from its native contaminants. Typically, a
monomeric
polypeptide is substantially pure when at least about 60 to 75% of a sample
exhibits a
single polypeptide backbone. Minor variants or chemical modifications
typically share
the same polypeptide sequence. Usually a substantially pure polypeptide will
comprise
over about 85 to 90% of a polypeptide sample, and preferably will be over
about 99%
pure. Normally, purity is measured on a polyacrylamide gel, with homogeneity
determined by staining. Alternatively, for certain purposes high resolution
will be
necessary and HPLC or a similar means for purification will be used. For most
purposes,
a simple chromatography column or polyacrylamide gel will be used to determine
purity.
[0029] The term "substantially free of naturally-associated host cell
components"
describes a polypeptide or other material which is separated from the native
contaminants
which accompany it in its natural host cell state. Thus, a polypeptide which
is chemically
synthesized or synthesized in a cellular system different from the host cell
from which it
naturally originates will be free from its naturally-associated host cell
components.
[0030] The terms "nucleic acid" or "polynucleotide" are used
interchangeably herein to
refer to a polymeric compound comprised of covalently linked subunits called
nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and
polydeoxyribonucleic
acid (DNA), both of which may be single-stranded or double-stranded. DNA
includes
but is not limited to cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-
synthetic DNA. DNA may be linear, circular, or supercoiled.

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[0031] A "nucleic acid molecule" refers to the phosphate ester polymeric
form of
ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules")
or
deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or
deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as
phosphorothioates and thioesters, in either single stranded form, or a double-
stranded
helix. D ouble stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
The term nucleic acid molecule, and in particular DNA or RNA molecule, refers
only to
the primary and secondary structure of the molecule, and does not limit it to
any
particular tertiary forms. Thus, this term includes, without limitation,
double-stranded
DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction
fragments),
plasmids, and chromosomes. In discussing the structure of particular double-
stranded
DNA molecules, sequences may be described herein according to the normal
convention
of giving only the sequence in the 5' to 3' direction along the non-
transcribed strand of
DNA (i.e., the strand having a sequence homologous to the mRNA). A
"recombinant
DNA molecule" is a DNA molecule that has undergone a molecular biological
manipulation.
[0032] The term "fragment" will be understood to mean a nucleotide
sequence of reduced
length relative to the reference nucleic acid and comprising, over the common
portion, a
nucleotide sequence identical to the reference nucleic acid. Such a nucleic
acid fragment
according to the invention may be, where appropriate, included in a larger
polynucleotide
of which it is a constituent. S uch fragments comprise, or alternatively
consist of,
oligonucleotides ranging in length from at least 6-1500 consecutive
nucleotides of a
nucleic acid according to the invention.
[0033] As used herein, an "isolated nucleic acid fragment" is a polymer of
RNA or DNA
that is single- or double-stranded, optionally containing synthetic, non-
natural or altered
nucleotide bases. An isolated nucleic acid fragment in the form of a polymer
of DNA
may be comprised of one or more segments of cDNA, genomic DNA or synthetic
DNA.
[0034] A "gene" refers to an assembly of nucleotides that encode an RNA
transcript or a
polypeptide, and includes cDNA and genomic DNA nucleic acids. "Gene" also
refers to
a nucleic acid fragment that expresses a specific protein or polypeptide,
including
regulatory sequences preceding (5' non-coding sequences) and following (3' non-
coding
sequences) the coding sequence. "Native gene" refers to a gene as found in
nature with

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its own regulatory sequences. "C himeric gene" refers to any gene that is not
a native
gene, comprising regulatory and/or coding sequences that are not found
together in
nature. A ccordingly, a chimeric gene may comprise regulatory sequences and
coding
sequences that are derived from different sources, or regulatory sequences and
coding
sequences derived from the same source, but arranged in a manner different
than that
found in nature. A chimeric gene may comprise coding sequences derived from
different
sources and/or regulatory sequences derived from different sources.
"Endogenous gene"
refers to a native gene in its natural location in the genome of an organism.
A "foreign"
gene or "heterologous" gene refers to a gene not normally found in the host
organism, but
that is introduced into the host organism by gene transfer. Foreign genes can
comprise
native genes inserted into a non-native organism, or chimeric genes. A
"transgene" is a
gene that has been introduced into the genome by a transformation procedure.
[0035] "Heterologous" DNA refers to DNA not naturally located in the cell,
or in a
chromosomal site of the cell. Preferably, the heterologous DNA includes a gene
foreign
to the cell.
[0036] The term "genome" includes chromosomal as well as mitochondrial,
chloroplast
and viral DNA or RNA.
[0037] A nucleic acid molecule is "hybridizable" to another nucleic acid
molecule, such
as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic
acid
molecule can anneal to the other nucleic acid molecule under the appropriate
conditions
of temperature and solution ionic strength (see Sambrook et al., 1989 infra).
Hybridization and washing conditions are well known and exemplified in
Sambrook, J.,
Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989),
particularly
Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference).
The
conditions of temperature and ionic strength determine the "stringency" of the
hybridization.
[0038] Stringency conditions can be adjusted to screen for moderately
similar fragments,
such as homologous sequences from distantly related organisms, to highly
similar
fragments, such as genes that duplicate functional enzymes from closely
related
organisms. F or preliminary screening for homologous nucleic acids, low
stringency
hybridization conditions, corresponding to a Tn, of 55 , can be used, e.g., 5x
SSC, 0.1%

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SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS).
Moderate
stringency hybridization conditions correspond to a higher Tõ, e.g., 40%
formamide, with
5x or 6x SCC. High stringency hybridization conditions correspond to the
highest Tõ,
e.g., 50% formamide, 5x or 6x SCC.
[0039] Hybridization requires that the two nucleic acids contain
complementary
sequences, although depending on t he stringency of the hybridization,
mismatches
between bases are possible. The term "complementary" is used to describe the
relationship between nucleotide bases that are capable of hybridizing to one
another. For
example, with respect to DNA, adenosine is complementary to thymine and
cytosine is
complementary to guanine. A ccordingly, the instant invention also includes
isolated
nucleic acid fragments that are complementary to the complete sequences as
disclosed or
used herein as well as those substantially similar nucleic acid sequences.
[0040] In a specific embodiment of the invention, polynucleotides are
detected by
employing hybridization conditions comprising a hybridization step at Tm of 55
C, and
utilizing conditions as set forth above. In certain embodiments, the Tm is 60
C, 63 C or
65 C.
[0041] Post-hybridization washes also determine stringency conditions. In
certain
embodiments the hybridization conditions use a series of washes starting with
6X SSC,
0.5% SDS at room temperature for 15 minutes (min), then repeated with 2X SSC,
0.5%
SDS at 45 C for 30 minutes, and then repeated twice with 0.2X SSC, 0.5% SDS at
50 C
for 30 minutes. A more stringent set of conditions uses higher temperatures in
which the
washes are identical to those above except for the temperature of the final
two 30 min
washes in 0.2X SSC, 0.5% SDS is increased to 60 C. A highly stringent set of
conditions
uses two final washes in 0.1X SSC, 0.1% SDS at 65 C.
[0042] The appropriate stringency for hybridizing nucleic acids depends on
the length of
the nucleic acids and the degree of complementation, variables well known in
the art.
The greater the degree of similarity or homology between two nucleotide
sequences, the
greater the value of Tm for hybrids of nucleic acids having those sequences.
The relative
stability (corresponding to higher Tm) of nucleic acid hybridizations
decreases in the
following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating Tm have been derived (see
Sambrook et
at., supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.,
oligonucleotides,

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the position of mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-
11.8).
[0043] Selectivity of hybridization exists when hybridization occurs which
is more
selective than total lack of specificity. Typically, selective hybridization
will occur when
there is at least about 55% homology over a stretch of at least about 14/25
nucleotides,
preferably at least about 65%, more preferably at least about 75%, and most
preferably at
least about 90%. See, Kanehisa, M. (1984), Nucleic Acids Res. 12:203-213,
which is
incorporated herein by reference. Stringent hybridization conditions will
typically include
salt concentrations of less than about 1 M, more usually less than about 500 m
M and
preferably less than about 200 mM. Temperature conditions will typically be
greater than
20 degrees Celsius, more usually greater than about 30 degrees Celsius and
preferably in
excess of about 37 degrees Celsius. A s other factors may significantly affect
the
stringency of hybridization, including, among others, base composition and
size of the
complementary strands, presence of organic solvents and extent of base
mismatching, the
combination of parameters is more important than the absolute measure of any
one.
[0044] In a specific embodiment of the invention, polynucleotides of the
invention are
detected by employing hybridization conditions comprising a hybridization step
in less
than 500 mM salt and at least 37 degrees Celsius, and a washing step in 2XSSPE
at least
63 degrees Celsius. In certain embodiment, the hybridization conditions
comprise less
than 200 mM salt and at least 37 degrees Celsius for the hybridization step.
In another
embodiment, the hybridization conditions comprise 2XSSPE and 63 degrees
Celsius for
both the hybridization and washing steps.
[0045] In one embodiment, the length for a hybridizable nucleic acid is at
least about 10
nucleotides. Preferable a minimum length for a hybridizable nucleic acid is at
least about
15 nucleotides; more preferably at least about 20 nucleotides; and even more
preferably
the length is at least 30 nucleotides. Furthermore, the skilled artisan will
recognize that
the temperature and wash solution salt concentration may be adjusted as
necessary
according to factors such as length of the probe.
[0046] The term "probe" refers to a single-stranded nucleic acid molecule
that can base
pair with a complementary single stranded target nucleic acid to form a double-
stranded
molecule.

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[0047] As used herein, the term "oligonucleotide" refers to a nucleic
acid, generally of at
least 18 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA
molecule,
a plasmid DNA or an mRNA molecule. 0 ligonucleotides can be labeled, e.g.,
with 32P-
nucleotides or nucleotides to which a label, such as biotin, has been
covalently
conjugated. A labeled oligonucleotide can be used as a probe to detect the
presence of a
nucleic acid. Oligonucleotides (one or both of which may be labeled) can be
used as PCR
primers, either for cloning full length or a fragment of a nucleic acid, or to
detect the
presence of a nucleic acid. An oligonucleotide can also be used to form a
triple helix with
a DNA molecule. Generally, oligonucleotides are prepared synthetically,
preferably on a
nucleic acid synthesizer. A ccordingly, oligonucleotides can be prepared with
non-
naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
[0048] A "primer" is an oligonucleotide that hybridizes to a target
nucleic acid sequence
to create a double stranded nucleic acid region that can serve as an
initiation point for
DNA synthesis under suitable conditions. Such primers may be used in a
polymerase
chain reaction.
[0049] "Polymerase chain reaction" is abbreviated PCR and means an in
vitro method for
enzymatically amplifying specific nucleic acid sequences. P CR involves a r
epetitive
series of temperature cycles with each cycle comprising three stages:
denaturation of the
template nucleic acid to separate the strands of the target molecule,
annealing a single
stranded PCR oligonucleotide primer to the template nucleic acid, and
extension of the
annealed primer(s) by DNA polymerase. PCR provides a means to detect the
presence of
the target molecule and, under quantitative or semi-quantitative conditions,
to determine
the relative amount of that target molecule within the starting pool of
nucleic acids.
[0050] "Reverse transcription-polymerase chain reaction" is abbreviated RT-
PCR and
means an in vitro method for enzymatically producing a target cDNA molecule or
molecules from an RNA molecule or molecules, followed by enzymatic
amplification of
a specific nucleic acid sequence or sequences within the target cDNA molecule
or
molecules as described above. RT-PCR also provides a means to detect the
presence of
the target molecule and, under quantitative or semi-quantitative conditions,
to determine
the relative amount of that target molecule within the starting pool of
nucleic acids.
[0051] A DNA "coding sequence" is a double-stranded DNA sequence that is
transcribed
and translated into a polypeptide in a cell in vitro or in vivo when placed
under the control

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of appropriate regulatory sequences. "Suitable regulatory sequences" refer to
nucleotide
sequences located upstream (5' non-coding sequences), within, or downstream
(3' non-
coding sequences) of a coding sequence, and which influence the transcription,
RNA
processing or stability, or translation of the associated coding sequence. R
egulatory
sequences may include, without limitation, promoters, translation leader
sequences,
introns, polyadenylation recognition sequences, RNA processing site, effector
binding
site and stem-loop structure. The boundaries of the coding sequence are
determined by a
start codon at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl)
terminus. A coding sequence can include, but is not limited to, prokaryotic
sequences,
cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If
the coding sequence is intended for expression in a eukaryotic cell, a
polyadenylation
signal and transcription termination sequence will usually be located 3' to
the coding
sequence.
[0052] "Open reading frame" is abbreviated ORF and means al ength of
nucleic acid
sequence, either DNA, cDNA or RNA, that comprises a translation start signal
or
initiation codon, such as an ATG or AUG, and a termination codon and can be
potentially
translated into a polypeptide sequence.
[0053] The term "head-to-head" is used herein to describe the orientation
of two
polynucleotide sequences in relation to each other. Two polynucleotides are
positioned in
a head-to-head orientation when the 5' end of the coding strand of one
polynucleotide is
adjacent to the 5' end of the coding strand of the other polynucleotide,
whereby the
direction of transcription of each polynucleotide proceeds away from the 5'
end of the
other polynucleotide. The term "head-to-head" may be abbreviated (5 ')-to-(5')
and may
also be indicated by the symbols ( ¨>) or
[0054] The term "tail-to-tail" is used herein to describe the orientation
of two
polynucleotide sequences in relation to each other. Two polynucleotides are
positioned in
a tail-to-tail orientation when the 3' end of the coding strand of one
polynucleotide is
adjacent to the 3' end of the coding strand of the other polynucleotide,
whereby the
direction of transcription of each polynucleotide proceeds toward the other
polynucleotide. The term "tail-to-tail" may be abbreviated (3')-to-(3') and
may also be
indicated by the symbols (¨> ) or

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[0055] The term "head-to-tail" is used herein to describe the orientation
of two
polynucleotide sequences in relation to each other. Two polynucleotides are
positioned in
a head-to-tail orientation when the 5' end of the coding strand of one
polynucleotide is
adjacent to the 3' end of the coding strand of the other polynucleotide,
whereby the
direction of transcription of each polynucleotide proceeds in the same
direction as that of
the other polynucleotide. The term "head-to-tail" may be abbreviated (5 ')-to-
(3') and
may also be indicated by the symbols (¨> ¨>) or (5'¨>3'5'¨>3').
[0056] The term "downstream" refers to a n ucleotide sequence that is
located 3' to
reference nucleotide sequence. In particular, downstream nucleotide sequences
generally
relate to sequences that follow the starting point of transcription. For
example, the
translation initiation codon of a gene is located downstream of the start site
of
transcription.
[0057] The term "upstream" refers to a nucleotide sequence that is located
5' to reference
nucleotide sequence. In particular, upstream nucleotide sequences generally
relate to
sequences that are located on t he 5' side of a coding sequence or starting
point of
transcription. For example, most promoters are located upstream of the start
site of
transcription.
[0058] The terms "restriction endonuclease" and "restriction enzyme" refer
to an enzyme
that binds and cuts within a specific nucleotide sequence within double
stranded DNA.
[0059] "Homologous recombination" refers to the insertion of a foreign DNA
sequence
into another DNA molecule, e.g., insertion of a vector in a chromosome.
Preferably, the
vector targets a specific chromosomal site for homologous recombination. F or
specific
homologous recombination, the vector will contain sufficiently long regions of
homology
to sequences of the chromosome to allow complementary binding and
incorporation of
the vector into the chromosome. Longer regions of homology, and greater
degrees of
sequence similarity, may increase the efficiency of homologous recombination.
[0060] Many methods known in the art may be used to propagate a
polynucleotide
according to the invention. 0 nce a suitable host system and growth conditions
are
established, recombinant expression vectors can be propagated and prepared in
quantity.
As described herein, the expression vectors which can be used include, but are
not limited
to, the following vectors or their derivatives: human or animal viruses such
as vaccinia
virus, adenovirus and adeno-associated virus (AAV); insect viruses such as
baculovirus;

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yeast vectors; bacteriophage vectors (e.g., lambda); and plasmid and cosmid
DNA
vectors, to name but a few.
[0061] A "vector" is any means for the cloning of and/or transfer of a
nucleic acid into a
host cell. A vector may be a replicon to which another DNA segment may be
attached so
as to bring about the replication of the attached segment. A "replicon" is any
genetic
element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an
autonomous unit of DNA replication in vivo, i.e., capable of replication under
its own
control. The term "vector" includes both viral and nonviral means for
introducing the
nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of
vectors known in the
art may be used to manipulate nucleic acids, incorporate response elements and
promoters
into genes, etc. Possible vectors include, for example but without limitation,
plasmids or
modified viruses including, for example bacteriophages such as lambda
derivatives, or
plasmids such as pBR322 or pUC plasmid derivatives, or the Bluescript vector.
For
example, the insertion of the DNA fragments corresponding to response elements
and
promoters into a suitable vector can be accomplished by ligating the
appropriate DNA
fragments into a chosen vector that has complementary cohesive termini.
Alternatively,
the ends of the DNA molecules may be enzymatically modified or any site may be
produced by ligating nucleotide sequences (linkers) into the DNA termini. Such
vectors
may be engineered to contain selectable marker genes that provide for the
selection of
cells that have incorporated the marker into the cellular genome. S uch
markers allow
identification and/or selection of host cells that incorporate and express the
proteins
encoded by the marker.
[0062] Viral vectors, and particularly retroviral vectors, have been used
in a wide variety
of gene delivery applications in cells, as well as living animal subjects.
Viral vectors that
can be used include but are not limited to retrovirus, adeno-associated virus
(AAV), pox,
baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus,
and
caulimovirus vectors. Non-viral vectors include, without limitation, plasmids,
liposomes,
electrically charged lipids (cytofectins), DNA-protein complexes, and
biopolymers. In
addition to a nucleic acid, a vector may also comprise one or more regulatory
regions,
and/or selectable markers useful in selecting, measuring, and monitoring
nucleic acid
transfer results (transfer to which tissues, duration of expression, etc.).

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[0063] The term "plasmid" refers to an extra chromosomal element often
carrying a gene
that is not part of the central metabolism of the cell, and usually in the
form of circular
double-stranded DNA molecules. S uch elements may be autonomously replicating
sequences, genome integrating sequences, phage or nucleotide sequences,
linear, circular,
or supercoiled, of a single- or double-stranded DNA or RNA, derived from any
source, in
which a number of nucleotide sequences have been joined or recombined into a
unique
construction which is capable of introducing a promoter fragment and DNA
sequence for
a selected gene product along with appropriate 3' untranslated sequence into a
cell.
[0064] A "cloning vector" is a "replicon", which is a unit length of a
nucleic acid,
preferably DNA, that replicates sequentially and which comprises an origin of
replication,
such as a plasmid, phage or cosmid, to which another nucleic acid segment may
be
attached so as to bring about the replication of the attached segment. Cloning
vectors
may be capable of replication in one cell type and expression in another
("shuttle
vector").
[0065] Vectors may be introduced into the desired host cells by methods
known in the
art, e.g., transfection, electroporation, microinjection, transduction, cell
fusion, DEAE
dextran, calcium phosphate precipitation, lipofection (lysosome fusion),
particle
bombardment, use of a gene gun, or a DNA vector transporter (see, e.g., Wu et
al., 1992,
J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624;
and
Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15,
1990).
[0066] A polynucleotide according to the invention can also be introduced
in vivo by
lipofection. F or the past decade, there has been increasing use of liposomes
for
encapsulation and transfection of nucleic acids in vitro. Synthetic cationic
lipids designed
to limit the difficulties and dangers encountered with liposome mediated
transfection can
be used to prepare liposomes for in vivo transfection of a g ene encoding a m
arker
(Felgner et al., 1987. PNAS 84:7413; Mackey, et al., 1988. Proc. Natl. Acad.
Sci. U.S.A.
85:8027-8031; and Ulmer et al., 1993. Science 259:1745-1748). The use of
cationic
lipids may promote encapsulation of negatively charged nucleic acids, and also
promote
fusion with negatively charged cell membranes (Felgner and Ringold, 1989.
Science
337:387-388). P articularly useful lipid compounds and compositions for
transfer of
nucleic acids are described in International Patent Publications W095/18863
and
W096/17823, and in U.S. Patent No. 5,459,127. T he use of lipofection to
introduce

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exogenous genes into the specific organs in vivo has certain practical
advantages.
Molecular targeting of liposomes to specific cells represents one area of
benefit. It is
clear that directing transfection to particular cell types would be
particularly preferred in a
tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the
brain. Lipids
may be chemically coupled to other molecules for the purpose of targeting
(Mackey, et
al., 1988, supra). T argeted peptides, e.g., hormones or neurotransmitters,
and proteins
such as antibodies, or non-peptide molecules could be coupled to liposomes
chemically.
[0067] Other molecules are also useful for facilitating transfection of a
nucleic acid in
vivo, such as a cationic oligopeptide (e.g., W095/21931), peptides derived
from DNA
binding proteins (e.g., W096/25508), or a cationic polymer (e.g., W095/21931).
[0068] It is also possible to introduce a vector in vivo as a naked DNA
plasmid (see U.S.
Patents 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated DNA delivery
approaches can also be used (Curiel et al., 1992. Hum. Gene Ther. 3:147-154;
and Wu
and Wu, 1987. J. Biol. Chem. 262:4429-4432).
[0069] The term "transfection" means the uptake of exogenous or
heterologous RNA or
DNA by a cell. A cell has been "transfected" by exogenous or heterologous RNA
or
DNA when such RNA or DNA has been introduced inside the cell. A cell has been
"transformed" by exogenous or heterologous RNA or DNA when the transfected RNA
or
DNA effects a p henotypic change. T he transforming RNA or DNA can be
integrated
(covalently linked) into chromosomal DNA making up the genome of the cell.
[0070] "Transformation" refers to the transfer of a nucleic acid molecule
into the genome
of a host organism, resulting in genetically stable inheritance. Host
organisms containing
the transformed nucleic acid molecule are referred to as "transgenic" or
"recombinant" or
"transformed" organisms.
[0071] The term "genetic region" will refer to a region of a nucleic acid
molecule or a
nucleotide sequence that comprises a gene encoding a polypeptide.
[0072] In addition, the recombinant vector comprising a polynucleotide
according to the
invention may include one or more origins for replication in the cellular
hosts in which
their amplification or their expression is sought, markers or selectable
markers.
[0073] The term "selectable marker" means an identifying factor, usually
an antibiotic or
chemical resistance gene, that is able to be selected for based upon the
marker gene's
effect, i.e., resistance to an antibiotic, resistance to a herbicide,
colorimetric markers,

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enzymes, fluorescent markers, and the like, wherein the effect is used to
track the
inheritance of a nucleic acid of interest and/or to identify a cell or
organism that has
inherited the nucleic acid of interest. Examples of selectable marker genes
known and
used in the art include, without limitation: genes providing resistance to
ampicillin,
streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide,
sulfonamide,
and the like; and genes that are used as phenotypic markers, i.e., anthocyanin
regulatory
genes, isopentanyl transferase gene, and the like. Selectable marker genes may
also be
considered reporter genes.
[0074] The term "reporter gene" means a nucleic acid encoding an
identifying factor that
is able to be identified based upon the reporter gene's effect, wherein the
effect is used to
track the inheritance of a nucleic acid of interest, to identify a cell or
organism that has
inherited the nucleic acid of interest, and/or to measure gene expression
induction or
transcription. Examples of reporter genes known and used in the art include,
without
limitation: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol
acetyltransferase (CAT), I3-galactosidase (LacZ), 13-glucuronidase (Gus), and
the like.
[0075] "Promoter" refers to a DNA sequence capable of controlling the
expression of a
coding sequence or functional RNA. In general, a coding sequence is located 3'
to a
promoter sequence. Promoters may be derived in their entirety from a native
gene, or be
composed of different elements derived from different promoters found in
nature, or even
comprise synthetic DNA segments. It is understood by those skilled in the art
that
different promoters may direct the expression of a gene in different tissues
or cell types,
or at different stages of development, or in response to different
environmental or
physiological conditions. Promoters that cause a gene to be expressed in most
cell types
at most times are commonly referred to as "constitutive promoters". Promoters
that cause
a gene to be expressed in a specific cell type are commonly referred to as
"cell-specific
promoters" or "tissue-specific promoters". Promoters that cause a gene to be
expressed at
a specific stage of development or cell differentiation are commonly referred
to as
"developmentally-specific promoters" or "cell differentiation-specific
promoters".
Promoters that are induced and cause a gene to be expressed following exposure
or
treatment of the cell with an agent, biological molecule, chemical, ligand,
light, or the like
that induces the promoter are commonly referred to as "inducible promoters" or
"regulatable promoters". It is further recognized that since in most cases the
exact

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boundaries of regulatory sequences have not been completely defined, DNA
fragments of
different lengths may have identical promoter activity.
[0076] A "promoter sequence" is a DNA regulatory region capable of binding
RNA
polymerase in a cell and initiating transcription of a downstream (3'
direction) coding
sequence. For purposes of defining the present invention, the promoter
sequence is
bounded at its 3' terminus by the transcription initiation site and extends
upstream (5'
direction) to include the minimum number of bases or elements necessary to
initiate
transcription at levels detectable above background. Within the promoter
sequence will
be found a transcription initiation site (conveniently defined for example, by
mapping
with nuclease Si), as well as protein binding domains (consensus sequences)
responsible
for the binding of RNA polymerase.
[0077] A coding sequence is "under the control" of transcriptional and
translational
control sequences in a cell when RNA polymerase transcribes the coding
sequence into
mRNA, which is then trans-RNA spliced (if the coding sequence contains
introns) and
translated into the protein encoded by the coding sequence.
[0078] "Transcriptional and translational control sequences" are DNA
regulatory
sequences, such as promoters, enhancers, terminators, and the like, that
provide for the
expression of a coding sequence in a host cell. In eukaryotic cells,
polyadenylation
signals are control sequences.
[0079] The term "response element" means one or more cis-acting DNA
elements which
confer responsiveness on a promoter mediated through interaction with the DNA-
binding
domains of the first chimeric gene. This DNA element may be either palindromic
(perfect
or imperfect) in its sequence or composed of sequence motifs or half sites
separated by a
variable number of nucleotides. The half sites can be similar or identical and
arranged as
either direct or inverted repeats or as a single half site or multimers of
adjacent half sites
in tandem. T he response element may comprise a minimal promoter isolated from
different organisms depending upon t he nature of the cell or organism into
which the
response element will be incorporated. T he DNA binding domain of the first
hybrid
protein binds, in the presence or absence of a ligand, to the DNA sequence of
a response
element to initiate or suppress transcription of downstream gene(s) under the
regulation of
this response element.

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[0080] The term "operably linked" refers to the association of nucleic
acid sequences on
a single nucleic acid fragment so that the function of one is affected by the
other. F or
example, a promoter is operably linked with a coding sequence when it is
capable of
affecting the expression of that coding sequence (i.e., that the coding
sequence is under
the transcriptional control of the promoter). Coding sequences can be operably
linked to
regulatory sequences in sense or antisense orientation.
[0081] The term "expression", as used herein, refers to the transcription
and stable
accumulation of sense (mRNA) or antisense RNA derived from a n ucleic acid or
polynucleotide. Expression may also refer to translation of mRNA into a
protein or
polypeptide.
[0082] The terms "cassette", "expression cassette" and "gene expression
cassette" refer to
a segment of DNA that can be inserted into a nucleic acid or polynucleotide at
specific
restriction sites or by homologous recombination. T he segment of DNA
comprises a
polynucleotide that encodes a polypeptide of interest, and the cassette and
restriction sites
are designed to ensure insertion of the cassette in the proper reading frame
for
transcription and translation. "Transformation cassette" refers to a s pecific
vector
comprising a polynucleotide that encodes a polypeptide of interest and having
elements in
addition to the polynucleotide that facilitate transformation of a particular
host cell.
Cassettes, expression cassettes, gene expression cassettes and transformation
cassettes of
the invention may also comprise elements that allow for enhanced expression of
a
polynucleotide encoding a polypeptide of interest in a host cell. T hese
elements may
include, but are not limited to: a promoter, a minimal promoter, an enhancer,
a response
element, a terminator sequence, a polyadenylation sequence, and the like.
[0083] The terms "modulate" and "modulates" mean to induce, reduce or
inhibit nucleic
acid or gene expression, resulting in the respective induction, reduction or
inhibition of
protein or polypeptide production.
[0084] The plasmids or vectors according to the invention may further
comprise at least
one promoter suitable for driving expression of a gene in a host cell. T he
term
"expression vector" means a vector, plasmid or vehicle designed to enable the
expression
of an inserted nucleic acid sequence following transformation into the host.
The cloned
gene, i.e., the inserted nucleic acid sequence, is usually placed under the
control of
control elements such as a promoter, a minimal promoter, an enhancer, or the
like.

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Initiation control regions or promoters, which are useful to drive expression
of a nucleic
acid in the desired host cell are numerous and familiar to those skilled in
the art.
Virtually any promoter capable of driving these genes is suitable for the
present invention
including but not limited to: viral promoters, bacterial promoters, animal
promoters,
mammalian promoters, synthetic promoters, constitutive promoters, tissue
specific
promoter, developmental specific promoters, inducible promoters, light
regulated
promoters; CYCl, HIS3, GAL], GAL4, GAL10, ADH1, PGK, PH05, GAPDH, ADC1,
TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful for
expression in
Saccharomyces); ACT] promoter (useful for expression in Pichia); b-lactamase,
lac, ara,
tet, trp, lPL, lPR, T7, tac, and trc promoters (useful for expression in
Escherichia coli);
light regulated-, seed specific-, pollen specific-, ovary specific-,
pathogenesis or disease
related-, cauliflower mosaic virus 35S, CMV 35S minimal, cassava vein mosaic
virus
(CsVMV), chlorophyll a/b binding protein, ribulose 1, 5-bisphosphate
carboxylase, shoot-
specific, root specific, chitinase, stress inducible, rice tungro bacilliform
virus, plant
super-promoter, potato leucine aminopeptidase, nitrate reductase, mannopine
synthase,
nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters (useful
for
expression in plant cells); animal and mammalian promoters known in the art
include, but
are not limited to, the SV40 early (SV40e) promoter region, the promoter
contained in the
3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of
the ElA or
major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus
(CMV)
early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter,
a
baculovirus IE1 promoter, an elongation factor 1 alpha (EF1) promoter, a
phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, an albumin
promoter, the regulatory sequences of the mouse metallothionein-L promoter and
transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, a-
actin,
tubulin and the like), the promoters of the intermediate filaments (desmin,
neurofilaments,
keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR,
CFTR or
factor VIII type, and the like), pathogenesis or disease related-promoters,
and promoters
that exhibit tissue specificity and have been utilized in transgenic animals,
such as the
elastase I gene control region which is active in pancreatic acinar cells;
insulin gene
control region active in pancreatic beta cells, immunoglobulin gene control
region active
in lymphoid cells, mouse mammary tumor virus control region active in
testicular, breast,

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lymphoid and mast cells; albumin gene, Apo Al and Apo All control regions
active in
liver, alpha-fetoprotein gene control region active in liver, alpha 1-
antitrypsin gene
control region active in the liver, beta-globin gene control region active in
myeloid cells,
myelin basic protein gene control region active in oligodendrocyte cells in
the brain,
myosin light chain-2 gene control region active in skeletal muscle, and
gonadotropic
releasing hormone gene control region active in the hypothalamus, pyruvate
kinase
promoter, villin promoter, promoter of the fatty acid binding intestinal
protein, promoter
of the smooth muscle cell a-actin, and the like. In addition, these expression
sequences
may be modified by addition of enhancer or regulatory sequences and the like.
[0085] Enhancers that may be used in embodiments of the invention include
but are not
limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation
factor 1
(EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.
[0086] Termination control regions, i.e., terminator or polyadenylation
sequences, may
also be derived from various genes native to the preferred hosts. 0 ptionally,
a
termination site may be unnecessary, however, it is most preferred if
included. In certain
embodiments of the invention, the termination control region may be comprise
or be
derived from a synthetic sequence, synthetic polyadenylation signal, an SV40
late
polyadenylation signal, an SV40 polyadenylation signal, a bovine growth
hormone
(BGH) polyadenylation signal, viral terminator sequences, or the like.
[0087] The terms "3' non-coding sequences" or "3' untranslated region
(UTR)" refer to
DNA sequences located downstream (3') of a coding sequence and may comprise
polyadenylation [poly(A)] recognition sequences and other sequences encoding
regulatory signals capable of affecting mRNA processing or gene expression. T
he
polyadenylation signal is usually characterized by affecting the addition of
polyadenylic
acid tracts to the 3' end of the mRNA precursor.
[0088] "Regulatory region" means a nucleic acid sequence that regulates
the expression
of a second nucleic acid sequence. A regulatory region may include sequences
which are
naturally responsible for expressing a p articular nucleic acid (a homologous
region) or
may include sequences of a different origin that are responsible for
expressing different
proteins or even synthetic proteins (a heterologous region). In particular,
the sequences
can be sequences of prokaryotic, eukaryotic, or viral genes or derived
sequences that
stimulate or repress transcription of a gene in a specific or non-specific
manner and in an

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inducible or non-inducible manner. Regulatory regions include, without
limitation,
origins of replication, RNA splice sites, promoters, enhancers,
transcriptional termination
sequences, and signal sequences which direct the polypeptide into the
secretory pathways
of the target cell.
[0089] A regulatory region from a "heterologous source" is a regulatory
region that is not
naturally associated with the expressed nucleic acid. Included among the
heterologous
regulatory regions are, without limitation, regulatory regions from a
different species,
regulatory regions from a different gene, hybrid regulatory sequences, and
regulatory
sequences which do not occur in nature, but which are designed by one having
ordinary
skill in the art.
[0090] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed
transcription of a DNA sequence. When the RNA transcript is a perfect
complementary
copy of the DNA sequence, it is referred to as the primary transcript or it
may be a RNA
sequence derived from post-transcriptional processing of the primary
transcript and is
referred to as the mature RNA. "Messenger RNA (mRNA)" refers to the RNA that
is
without introns and that can be translated into protein by the cell. "cDNA"
refers to a
double-stranded DNA that is complementary to and derived from mRNA. "Sense"
RNA
refers to RNA transcript that includes the mRNA and so can be translated into
protein by
the cell. "Antisense RNA" refers to a RNA transcript that is complementary to
all or part
of a target primary transcript or mRNA and that blocks the expression of a
target gene.
The complementarity of an antisense RNA may be with any part of the specific
gene
transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, or
the coding
sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA
that is not translated yet has an effect on cellular processes.
[0091] A "polypeptide" is a polymeric compound comprised of covalently
linked amino
acid residues. Amino acids have the following general structure:
H
R¨C¨COOH
NH2
[0092] Amino acids are classified into seven groups on the basis of the
side chain R: (1)
aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3)
side chains

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containing sulfur atoms, (4) side chains containing an acidic or amide group,
(5) side
chains containing a basic group, (6) side chains containing an aromatic ring,
and (7)
proline, an imino acid in which the side chain is fused to the amino group. A
polypeptide
of the invention preferably comprises at least about 14 amino acids.
[0093] An "isolated polypeptide" or "isolated protein" is a polypeptide or
protein that is
substantially free of those compounds that are normally associated therewith
in its natural
state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates,
lipids). "Isolated"
is not meant to exclude artificial or synthetic mixtures with other compounds,
or the
presence of impurities which do not interfere with biological activity, and
which may be
present, for example, due to incomplete purification, addition of stabilizers,
or
compounding into a pharmaceutically acceptable preparation.
[0094] A "fragment" of a polypeptide according to the invention will be
understood to
mean a p olypeptide whose amino acid sequence is shorter than that of the
reference
polypeptide and which comprises, over the entire portion with these reference
polypeptides, an identical amino acid sequence. Such fragments may, where
appropriate,
be included in a larger polypeptide of which they are a part. S uch fragments
of a
polypeptide according to the invention may have a length of at least 2-300
amino acids.
[0095] A "heterologous protein" refers to a protein not naturally produced
in the cell.
[0096] A "mature protein" refers to a post-translationally processed
polypeptide; i.e., one
from which any pre- or propeptides present in the primary translation product
have been
removed. "Precursor" protein refers to the primary product of translation of
mRNA; i.e.,
with pre- and propeptides still present. Pre- and propeptides may be but are
not limited to
intracellular localization signals.
[0097] The term "signal peptide" refers to an amino terminal polypeptide
preceding the
secreted mature protein. The signal peptide is cleaved from and is therefore
not present in
the mature protein. S ignal peptides have the function of directing and
translocating
secreted proteins across cell membranes. S ignal peptide is also referred to
as signal
protein.
[0098] A "signal sequence" is included at the beginning of the coding
sequence of a
protein to be expressed on the surface of a cell. This sequence encodes a
signal peptide,
N-terminal to the mature polypeptide, that directs the host cell to
translocate the
polypeptide. The term "translocation signal sequence" is used herein to refer
to this sort

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of signal sequence. Translocation signal sequences can be found associated
with a variety
of proteins native to eukaryotes and prokaryotes, and are often functional in
both types of
organisms.
[0099] The term "homology" refers to the percent of identity between two
polynucleotide
or two polypeptide moieties. The correspondence between the sequence from one
moiety
to another can be determined by techniques known to the art. F or example,
homology
can be determined by a direct comparison of the sequence information between
two
polypeptide molecules by aligning the sequence information and using readily
available
computer programs. Alternatively, homology can be determined by hybridization
of
polynucleotides under conditions that form stable duplexes between homologous
regions,
followed by digestion with single-stranded-specific nuclease(s) and size
determination of
the digested fragments.
[0100] As used herein, the term "homologous" in all its grammatical forms
and spelling
variations refers to the relationship between proteins that possess a "common
evolutionary origin," including proteins from superfamilies (e.g., the
immunoglobulin
superfamily) and homologous proteins from different species (e.g., myosin
light chain,
etc.) (Reeck et al., 1987, Cell 50:667.). Such proteins (and their encoding
genes) have
sequence homology, as reflected by their high degree of sequence similarity.
However, in
common usage and in the instant application, the term "homologous," when
modified
with an adverb such as "highly," may refer to sequence similarity and not a
common
evolutionary origin.
[0101] Accordingly, the term "sequence similarity" in all its grammatical
forms refers to
the degree of identity or correspondence between nucleic acid or amino acid
sequences of
proteins that may or may not share a common evolutionary origin (see Reeck et
al., 1987,
Cell 50:667).
[0102] In a specific embodiment, two DNA sequences are "substantially
homologous" or
"substantially similar" when at least about 50% (preferably at least about
75%, and most
preferably at least about 90 or 95%) of the nucleotides match over the defined
length of
the DNA sequences.
[0103] Sequences that are substantially homologous can be identified by
comparing the
sequences using standard software available in sequence data banks, or in a
Southern
hybridization experiment under, for example, stringent conditions as defined
for that

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particular system. Defining appropriate hybridization conditions is within the
skill of the
art. See, e.g., Sambrook et at., 1989, supra.
[0104] As used herein, "substantially similar" refers to nucleic acid
fragments wherein
changes in one or more nucleotide bases results in substitution of one or more
amino
acids, but do not affect the functional properties of the protein encoded by
the DNA
sequence. "Substantially similar" also refers to nucleic acid fragments
wherein changes
in one or more nucleotide bases does not affect the ability of the nucleic
acid fragment to
mediate alteration of gene expression by antisense or co-suppression
technology.
"Substantially similar" also refers to modifications of the nucleic acid
fragments of the
instant invention such as deletion or insertion of one or more nucleotide
bases that do not
substantially affect the functional properties of the resulting transcript. It
is therefore
understood that the invention encompasses more than the specific exemplary
sequences.
Each of the proposed modifications is well within the routine skill in the
art, as is
determination of retention of biological activity of the encoded products.
[0105] Moreover, the skilled artisan recognizes that substantially similar
sequences
encompassed by this invention are also defined by their ability to hybridize,
under
stringent conditions (0.1X SSC, 0.1% SDS, 65 C and washed with 2X SSC, 0.1%
SDS
followed by 0.1X SSC, 0.1% SDS), with the sequences exemplified herein.
Substantially
similar nucleic acid fragments of the instant invention are those nucleic acid
fragments
whose DNA sequences are at least 70% identical to the DNA sequence of the
nucleic acid
fragments reported herein. Substantially similar nucleic acid fragments of the
instant
invention include those nucleic acid fragments whose DNA sequences are at
least 80%
identical to the DNA sequence of the nucleic acid fragments reported herein.
In certain
embodiments nucleic acid fragments are at least 90% identical, at least 95%
identical, at
least 97% identical, at least 98% identical, or at least 99% identical to the
DNA sequence
of the nucleic acid fragments reported herein. In certain embodiments,
substantially
similar nucleotide sequences of the invention can encode any polypeptide
sequences
described in the present application (e.g., scIL-12 polypeptides) despite any
differences in
nucleotide sequences present in comparison to specific polynucleotide
sequences
described herein.
[0106] Two amino acid sequences are "substantially homologous" or
"substantially
similar" when greater than about 40% of the amino acids are identical, or
greater than

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60% are similar (functionally identical). Preferably, the similar or
homologous sequences
are identified by alignment using, for example, the GCG (Genetics Computer
Group,
Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup
program.
[0107] The term "corresponding to" is used herein to refer to similar
or homologous
sequences, whether the exact position is identical or different from the
molecule to which
the similarity or homology is measured. A nucleic acid or amino acid sequence
alignment may include spaces. Thus, the term "corresponding to" refers to the
sequence
similarity, and not the numbering of the amino acid residues or nucleotide
bases.
[0108] A "substantial portion" of an amino acid or nucleotide sequence
comprises
enough of the amino acid sequence of a polypeptide or the nucleotide sequence
of a gene
to putatively identify that polypeptide or gene, either by manual evaluation
of the
sequence by one skilled in the art, or by computer-automated sequence
comparison and
identification using algorithms such as BLAST (Basic Local Alignment Search
Tool;
Altschul, S. F., et al., (1993) J. Mol. Biol.
215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous
amino acids or thirty or more nucleotides is necessary in order to putatively
identify a
polypeptide or nucleic acid sequence as homologous to a known protein or gene.
Moreover, with respect to nucleotide sequences, gene specific oligonucleotide
probes
comprising 20-30 contiguous nucleotides may be used in sequence-dependent
methods of
gene identification (e.g., Southern hybridization) and isolation (e.g., in
situ hybridization
of bacterial colonies or bacteriophage plaques). In addition, short
oligonucleotides of
12-15 bases may be used as amplification primers in PCR in order to obtain a
particular
nucleic acid fragment comprising the primers. Accordingly, a "substantial
portion" of a
nucleotide sequence comprises enough of the sequence to specifically identify
and/or
isolate a nucleic acid fragment comprising the sequence.
[0109] The term "percent identity", as known in the art, is a
relationship between two or
more polypeptide sequences or two or more polynucleotide sequences, as
determined by
comparing the sequences. In the art, "identity" also means the degree of
sequence
relatedness between polypeptide or polynucleotide sequences, as the case may
be, as
determined by the match between strings of such sequences. "Identity" and
"similarity"
can be readily calculated by known methods, including but not limited to those
described
in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University
Press, New

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York (1988); Biocomputing: Informatics and G enome Projects (Smith, D. W.,
ed.)
Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I
(Griffin,
A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence
Analysis in
Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence
Analysis
Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).
Preferred methods to determine identity are designed to give the best match
between the
sequences tested. Methods to determine identity and similarity are codified in
publicly
available computer programs. Sequence alignments and percent identity
calculations may
be performed using the Megalign program of the LASERGENE bioinformatics
computing suite (DNASTAR Inc., Madison, WI). Multiple alignment of the
sequences
may be performed using the Clustal method of alignment (Higgins and Sharp
(1989)
CABIOS. 5:151-153) with the default parameters (GAP PENALTY= 10, GAP LENGTH
PENALTY= 10). Default parameters for pairwise alignments using the Clustal
method
may be selected: KTUPLE 1, GAP PENALTY= 3, WINDOW= 5 and DIAGONALS
SAVED=5.
[0110] The term "sequence analysis software" refers to any computer
algorithm or
software program that is useful for the analysis of nucleotide or amino acid
sequences.
"Sequence analysis software" may be commercially available or independently
developed. Typical sequence analysis software will include but is not limited
to the GCG
suite of programs (Wisconsin Package Version 9.0, G enetics Computer Group
(GCG),
Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-
410
(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA).
Within the context of this application it will be understood that where
sequence analysis
software is used for analysis, that the results of the analysis will be based
on the "default
values" of the program referenced, unless otherwise specified. As used herein
"default
values" will mean any set of values or parameters which originally load with
the software
when first initialized.
[0111] "Synthetic genes" can be assembled from oligonucleotide building
blocks that are
chemically synthesized using procedures known to those skilled in the art. T
hese
building blocks are ligated and annealed to form gene segments that are then
enzymatically assembled to construct the entire gene. "C hemically
synthesized", as
related to a sequence of DNA, means that the component nucleotides were
assembled

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in vitro. Manual chemical synthesis of DNA may be accomplished using well
established
procedures, or automated chemical synthesis can be performed using one of a
number of
commercially available machines. A ccordingly, the genes can be tailored for
optimal
gene expression based on optimization of nucleotide sequence to reflect the
codon bias of
the host cell. The skilled artisan appreciates the likelihood of successful
gene expression
if codon usage is biased towards those codons favored by the host. D
etermination of
preferred codons can be based on a survey of genes derived from the host cell
where
sequence information is available. Alternatively, or in addition to
optimization to reflect
codon bias, optimization can also include optimization of nucleotide sequence
based on
specific host cells wherein optimization is performed to maximize
transcription rate or
quantity, transcript half-life, and translation rate or quantity. Such
optimization can be
performed through empirical determinations based on specific host cell.
[0112] The term "gene switch" refers to the combination of a response
element associated
with a promoter, and a ligand-dependent transcription factor-based system
which, in the
presence of one or more ligands, modulates the expression of a gene with which
the
response element and promoter are operably associated. The term "a
polynucleotide
encoding a gene switch" refers to the combination of a response element
associated with a
promoter, and a polynucleotide encoding a ligand-dependent transcription
factor-based
system which, in the presence of one or more ligands, modulates the expression
of a gene
with which the response element and promoter are operably associated.
[0113] The terms "IL-12 activity" and "IL-12 biological activity" refer to
any of the well-
known bioactivities of IL-12, and include, without limitation, stimulating
differentiation
of naive T cells into Thl cells, stimulating growth and function of T cells,
stimulating
production of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha
(TNF-
alpha) from T-cells and natural killer (NK) cells, stimulating reduction of IL-
4 mediated
suppression of IFN-gamma, stimulating enhancement of the cytotoxic activity of
NK cells
and CD8 ' cytotoxic T lymphocytes, stimulating expression of IL-12R-betal and
IL-12R-
beta2, facilitating the presentation of tumor antigens through the
upregulation of MHC I
and II molecules, and stimulating anti-angiogenic activity. Exemplary assays
for IL-12
activity include the Gamma Interferon Induction Assay (see Example 3, and US
Patent
5,457,038). Additional assays are known in the art, such as, but not limited
to, NK Cell
Spontaneous Cytotoxicity Assays, ADCC Assays, Co-Mitogenic Effect Assays, and
GM-

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CSF Induction Assays (e.g., as disclosed in Example 8 of US Patent 5,457,038,
incorporated herein by reference).
[0114] In a preferred embodiment, scIL-12 polypeptides of the invention
retain at least
one IL-12 biological activity. In certain embodiments, scIL-12 polypeptides of
the
invention retain more than one IL-12 biological activity. In certain
embodiments, scIL-12
polypeptides of the invention retain at least one, at least two, at least
three, at least four, at
least five or at least six of the above-referenced IL-12 biological
activities. In certain
embodiments, the IL-12 biological activity of scIL-12 polypeptides of the
present
invention is compared to (assayed against) the heterodimeric p3 5/p40 (wild-
type) form of
IL-12. In certain embodiments, scIL-12 polypeptides of the invention retain at
least about
50%, at least about 75%, at least about 85%, at least about 90%, at least
about 100%, at
least 50%, at least 75%, at least 85%, at least 90%, at least 100%, or more of
the
biological activity of IL-12 compared to the heterodimeric p35/p40 (wild-type)
form of
IL-12.
[0115] As used herein, the terms "treating" or "treatment" of a disease
refer to executing a
protocol, which may include administering one or more drugs or in vitro
engineered cells
to a mammal (human or non-human), in an effort to alleviate signs or symptoms
of the
disease. Thus, "treating" or "treatment" should not necessarily be construed
to require
complete alleviation of signs or symptoms, does not require a cure, and
specifically
includes protocols that have only marginal effect on the subject.
[0116] As used herein, "immune cells" include dendritic cells,
macrophages, neurophils,
mast cells, eosinophils, basophils, natural killer cells and lymphocytes
(e.g., B and T
cells).
[0117] As used herein, the term "stem cells" includes embryonic stem
cells, adult stem
cells and induced pluripotent stem cells. Stem cells can be obtained from any
appropriate
source, including bone marrow, adipose tissue, and blood (including, but not
limited to,
umbilical cord blood and menstrual blood). Examples of stem cells include, but
are not
limited to, mesenchymal stem cells and hematopoietic stem cells.
[0118] As used herein, the terms "dendritic cells" and "DC" are
interchangeably used.
Likewise, the terms "Natural Killer Cells" and "NK cells" are interchangeably
used.

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Polynucleotides Encoding Single Chain IL-12 Polypeptides
[0119]
The present invention provides novel polynucleotides encoding a single chain
interleukin-12 (scIL-12) polypeptide of the invention, including full length
and mature
scIL-12 polypeptides.
[0120] In accordance with specific embodiments of the present
invention, nucleic acid
sequences encoding novel scIL-12 polypeptides are provided. Specifically, the
invention
provides polynucleotides encoding a scIL-12 polypeptide comprising, from N- to
C-
terminus:
(i) a first IL-12 p40 domain (p4ON),
(ii) an optional first peptide linker,
(iii) an IL-12 p35 domain,
(iv) an optional second peptide linker, and
(v) a second IL-12 p40 domain (p40C).
[0121]
In certain embodiments, the first IL-12 p40 domain (also referred to herein as
p4ON) encoded by polynucleotides of the invention is an N-terminal fragment of
an IL-12
p40 subunit. IL-12 p40 polynucleotides for use in the invention include the
human IL-12
p40 nucleic acid sequence of SEQ ID NO: 1 and the murine IL-12 p40 nucleic
acid
sequence of SEQ ID NO: 5. A dditional, non-limiting examples of
polynucleotides
encoding IL-12 p40 subunits are available in public sequence databases,
including but not
limited to Genbank Accession Nos. AF180563.1 (human), NM 002187.2 (human),
NG 009618.1 (human), NM 001077413.1 (cat), AF091134.1 (dog), NM 008352.2
(mouse), NM 001159424.1 (mouse), and NM 008351.2 (mouse).
[0122]
N-terminal fragments of IL-12 p40 encoded by polynucleotides of the invention
and
suitable as a first IL-12 p40 dom am n (p4ON) include, but are not limited to,
polypeptides comprising, or alternatively consisting of, amino acids 1 to 288,
1 to 289, 1
to 290, 1 to 291, 1 to 292, 1 to 293, 1 to 294, 1 to 295, 1 to 296, 1 to 297,
and 1 to 298 of
SEQ ID NO: 2. A preferred N-terminal fragment of IL-12 p40 encoded by
polynucleotides of the invention and suitable as a first IL-12 p40 d omain
(p4ON)
comprises, or alternatively consists of, amino acids 1 to 293 of SEQ ID NO: 2.
[0123] N-terminal fragments of IL-12 p40 encoded by polynucleotides of
the invention
and suitable as a first IL-12 p40 dom am n (p4ON) may lack a s ignal sequence.
It is
understood that the specific cleavage site of a signal peptide may vary by 1,
2, 3 or more

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residues. A ccordingly, in additional embodiments the first IL-12 p40 domain
(p4ON)
encoded by polynucleotides of the invention comprises, or alternatively
consists of, a
fragment of SEQ ID NO: 2 beginning with residue 18, 19, 20, 21, 22, 23, 24,
25, 26, 27 or
28 of SEQ ID NO: 2 and ending with residue 288, 289, 290, 291, 292, 293, 294,
295, 296,
297, or 298 of SEQ ID NO: 2. In on e embodiment, a first IL-12 p40 domain
(p4ON)
encoded by polynucleotides of the invention comprises, or alternatively
consists of,
amino acid residues 23 to 293 of SEQ ID NO: 2.
[0124] The optional first peptide linker (ii) may be any suitable
peptide linker that allows
folding of the scIL-12 polypeptide into a functional protein. In certain
embodiments, the
optional first peptide linker encoded by polynucleotides of the invention
consists of 10 or
fewer amino acids. In specific embodiments, the first peptide linker consists
of 1, 2, 3, 4,
5, 6, 7, 8, 9, o r 10 amino acids. In specific embodiments, the first peptide
linker
comprises any sequence and combination of one or more amino acids selected
from:
Glycine (Gly); Serine (Ser); Alanine (Ala); Threonine (Thr); and, Proline
(Pro). In a
preferred embodiment, the first peptide linker is selected from the peptides
Thr-Pro-Ser
(SEQ ID NO: 41) and Ser-Gly-Pro-Ala-Pro (SEQ ID NO: 42), and peptides with one
amino acid substitution in Thr-Pro-Ser (SEQ ID NO: 41) and Ser-Gly-Pro-Ala-Pro
(SEQ
ID NO: 42). In certain embodiments the first peptide linker is absent.
[0125] In certain embodiments, the IL-12 p35 domain (iii) encoded by
polynucleotides of
the invention is a mature IL-12 p35 s ubunit, lacking a signal peptide. IL-12
p35
polynucleotides for use in the invention include the human IL-12 p35 nucleic
acid
sequence of SEQ ID NO: 3 and the murine IL-12 p35 nucleic acid sequence of SEQ
ID
NO: 7. Additional, non-limiting examples of polynucleotides encoding IL-12 p35
subunits are available in public sequence databases, including but not limited
to
AF101062 .1 (human), NM 000882.3 (human), NG 033022.1
(human),
NM 001159424.1 (mouse), NM 008351.2 (mouse), NM 001009833 (cat),
NM 001082511.1 (horse), NM 001003293.1 (dog).
[0126] It is understood that the specific cleavage site of a signal
peptide may vary by 1, 2,
3 or more residues. Accordingly, IL-12 p35 domains encoded by polynucleotides
of the
invention include the predicted mature sequence comprising, or alternatively
consisting
of, residues 57 to 253 of SEQ ID NO: 4 as well as mature sequences comprising,
or

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alternatively consisting of, amino acids 52 to 253, 53 to 253, 54 to 253, 55
to 253, 56 to
253, 58 to 253, 59 to 253, 60 to 253, 61 to 263 and 62 to 253 of SEQ ID NO: 4.
[0127] Suitable IL-12 p35 domains encoded by polynucleotides of the
invention may be
truncated at the C-terminus by one or more amino acid residues. Therefore, in
additional
embodiments the IL-12 p35 dom am n encoded by polynucleotides of the invention
comprise, or alternatively consist of, a fragment of SEQ ID NO: 4 beginning
with residue
52, 53, 54, 55, 56, 57, 5 8, 59, 60, 61, or 62 of SEQ ID NO: 4 and ending with
residue
247, 248, 249, 250, 251, 252, or 253 of SEQ ID NO: 4.
[0128] The optional second peptide linker (iv) may be any suitable
peptide linker that
allows folding of the scIL-12 polypeptide into a functional protein. In
certain
embodiments, the optional second peptide linker encoded by polynucleotides of
the
invention consists of 10 or fewer amino acids. In specific embodiments, the
second
peptide linker consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 a mino acids.
In specific
embodiments, the second peptide linker comprises any sequence and combination
of one
or more amino acids selected from: Glycine (Gly); Serine (Ser); Alanine (Ala);
Threonine
(Thr); and, Proline (Pro). In a preferred embodiment, the second peptide
linker is
selected from the peptides Thr-Pro-Ser (SEQ ID NO: 41) and Ser-Gly-Pro-Ala-Pro
(SEQ
ID NO: 42), and peptides with one amino acid substitution in Thr-Pro-Ser (SEQ
ID NO:
41) and Ser-Gly-Pro-Ala-Pro (SEQ ID NO: 42). In certain embodiments the second
peptide linker is absent. In a preferred embodiment, the first and second
peptide linkers
consist of 10, 9, 8, 7 or fewer amino acid residues combined.
[0129] In certain embodiments, the second IL-12 p40 domain (also
referred to herein as
p40C) encoded by polynucleotides of the invention is a C-terminal fragment of
an IL-12
p40 subunit. C-terminal fragments of IL-12 p40 encoded by polynucleotides of
the
invention and suitable as a second IL-12 p40 domain (p40C) comprise, or
alternatively
consist of, amino acids 289 to 328, 290 to 328, 291 to 328, 292 to 328, 293 to
328, 294 to
328, 295 to 328, 296 to 328, 297 to 328, 298 to 328, and 299 to 328 of SEQ ID
NO: 2.
[0130] Suitable second IL-12 p40 domains (p40C) encoded by
polynucleotides of the
invention may be truncated at the C-terminus by one or more amino acid
residues.
Accordingly, in additional embodiments the second IL-12 p40 domain (p40C)
encoded by
polynucleotides of the invention comprises, or alternatively consists of, a
fragment of
SEQ ID NO: 2 beginning with residue 289, 290, 291, 292, 293, 294, 295, 296,
297, 298,

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or 299 of SEQ ID NO: 2 and ending with residue 322, 323, 324, 325, 326, 327,
or 328 of
SEQ ID NO: 2.
[0131] The full-length sequence of a polynucleotide encoding a preferred
scIL-12
polypeptide of the invention is presented herein as SEQ ID NO: 9. The full-
length
sequence encodes a predicted signal peptide at nucleic acids 1 to 66 of SEQ ID
NO: 9,
and a mature scIL-12 polypeptide at nucleic acids 67 to 1599 of SEQ ID NO: 9.
[0132] Thus, a first subject of the invention relates to an isolated
polynucleotide encoding
a novel scIL-12 polypeptide. In a specific embodiment, the isolated
polynucleotide
comprises a nucleic acid sequence selected from the group consisting of SEQ ID
NO: 9
and nucleic acids 67 to 1599 of SEQ ID NO: 9. In a specific embodiment, the
isolated
polynucleotide further comprises a region permitting expression of the
polypeptide in a
host cell.
[0133] The present invention also relates to an isolated polynucleotide
encoding a scIL-
12 polypeptide comprising an amino acid sequence selected from the group
consisting of
SEQ ID NO: 10 and amino acids 23 to 533 of SEQ ID NO: 10.
[0134] The invention also provides polynucleotides encoding variants of
the scIL-12
polypeptides of the invention. In a preferred embodiment the polynucleotides
of the
invention encode a scIL-12 variant polypeptide at least 80%, at least 85%, at
least 90%, at
least 95%, at least 97%, at least 98%, or at least 99% identical to the full-
length or mature
amino acid sequence of SEQ ID NO: 10, where the variant polypeptide exhibits
at least
one IL-12 activity, such as induction of IFN-gamma secretion from NK cells.
Such IL-12
activities are readily determined using assays known in the art, such as the
assays
described in Example 8 of US Patent 5,457,038, which is incorporated herein by
reference.
[0135] Due to the degeneracy of nucleotide coding sequences, other
polynucleotides that
encode substantially the same amino acid sequence as a scIL-12 polynucleotide
disclosed
herein, including an amino acid sequence that contains a single amino acid
variant, may
be used in the practice of the present invention. T hese include but are not
limited to
allelic genes, homologous genes from other species, and nucleotide sequences
comprising
all or portions of a scIL-12 polynucleotide that are altered by the
substitution of different
codons that encode the same amino acid residue within the sequence, thus
producing a
silent change. Likewise, the scIL-12 derivatives of the invention include, but
are not

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limited to, those comprising, as a primary amino acid sequence, all or part of
the amino
acid sequence of a scIL-12 polypeptide including altered sequences in which
functionally
equivalent amino acid residues are substituted for residues within the
sequence resulting
in a conservative amino acid substitution. For example, one or more amino acid
residues
within the sequence can be substituted by another amino acid of a similar
polarity, which
acts as a functional equivalent, resulting in a silent alteration. Substitutes
for an amino
acid within the sequence may be selected from other members of the class to
which the
amino acid belongs. F or example, the nonpolar (hydrophobic) amino acids
include
alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and
methionine.
Amino acids containing aromatic ring structures are phenylalanine, tryptophan,
and
tyrosine. T he polar neutral amino acids include glycine, serine, threonine,
cysteine,
tyrosine, asparagine, and glutamine. The positively charged (basic) amino
acids include
arginine, lysine and histidine. T he negatively charged (acidic) amino acids
include
aspartic acid and glutamic acid. Such alterations can be produced by various
methods
known in the art (see Sambrook et al., 1989, infra) and are not expected to
affect apparent
molecular weight as determined by polyacrylamide gel electrophoresis, or
isoelectric
point.
[0136] The present invention also relates to an isolated scIL-12
polypeptide encoded by a
polynucleotide according to the invention.
Single Chain IL-12 Polypeptides
[0137] The present invention provides novel scIL-12 polypeptides,
including full length
and mature scIL-12 polypeptides.
[0138] Thus, the invention relates to isolated scIL-12 polypeptides. In a
s pecific
embodiment, the invention provides a scIL-12 polypeptide comprising, from N-
to C-
terminus:
(i) a first IL-12 p40 domain (p4ON),
(ii) an optional first peptide linker,
(iii) an IL-12 p35 domain,
(iv) an optional second peptide linker, and
(v) a second IL-12 p40 domain (p40C).
[0139] In certain embodiments, the first IL-12 p40 do main (p4ON) is an N-
terminal
fragment of an IL-12 p40 subunit. IL-12 p40 polypeptides for use in the
invention

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include the human IL-12 p40 amino acid sequence of SEQ ID NO: 2 and the murine
IL-
12 p40 amino acid sequence of SEQ ID NO: 6. Additional, non-limiting examples
of IL-
12 p40 subunits are available in public sequence databases, including but not
limited to
Genbank Accession Nos. P29460.1 (human), AAD56386.1 (human), NP 005526.1
(human), NP 714912.1 (human), Q28268.1 (dog), NP 001003292.1 (dog), NP
032378.1
(mouse), NP 001152896.1 (mouse), NP 032377.1 (mouse).
[0140] N-terminal fragments of IL-12 p40 suitable as a first IL-12 p40
domain (p4ON)
include, but are not limited to, polypeptides comprising, or alternatively
consisting of,
amino acids 1 to 288, 1 to 289, 1 to 290, 1 to 291, 1 to 292, 1 to 293, 1 to
294, 1 to 295, 1
to 296, 1 to 297, and 1 to 298 of SEQ ID NO: 2. A preferred first IL-12 p40
domain
(p4ON) comprises, or alternatively consists of, amino acids 1 to 293 of SEQ ID
NO: 2.
[0141] N-terminal fragments of IL-12 p40 suitable as a first IL-12 p40
domain (p4ON)
may lack a signal sequence. Therefore, in additional embodiments the first IL-
12 p40
domain (p4ON) comprises, or alternatively consists of, a fragment of SEQ ID
NO: 2
beginning with residue 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 of SEQ ID
NO: 2 and
ending with residue 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, or 298.
In one
embodiment, the first IL-12 p40 domain (p4ON) comprises, or alternatively
consists of,
amino acid residues 23 to 293 of SEQ ID NO: 2.
[0142] The optional first peptide linker (ii) may be any suitable
peptide linker that allows
folding of the scIL-12 polypeptide into a functional protein. In certain
embodiments, the
optional first peptide linker consists of 10 or fewer amino acids.
In specific
embodiments, the first peptide linker consists of 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 amino acids.
In specific embodiments, the first peptide linker comprises any sequence and
combination
of one or more amino acids selected from: Glycine (Gly); Serine (Ser); Alanine
(Ala);
Threonine (Thr); and, Proline (Pro). In a preferred embodiment, the first
peptide linker is
selected from the peptides Thr-Pro-Ser (SEQ ID NO: 41) and Ser-Gly-Pro-Ala-Pro
(SEQ
ID NO: 42), and peptides with one amino acid substitution in Thr-Pro-Ser (SEQ
ID NO:
41) and Ser-Gly-Pro-Ala-Pro (SEQ ID NO: 42). In certain embodiments the first
peptide
linker is absent.
[0143] In certain embodiments, the IL-12 p35 domain (iii) is a mature
IL-12 p35 subunit,
lacking a signal peptide. IL-12 p35 pol ypeptides for use in the invention
include the
human IL-12 p35 amino acid sequence of SEQ ID NO: 4 and the murine IL-12p35
amino

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acid sequence of SEQ ID NO: 8. A dditional, non-limiting examples of IL-12 p35
subunits are available in public sequence databases, including but not limited
to Genbank
Accession Nos. AAB32758.1 (cat), NP 001003293 (dog), NP 001075980.1 (horse),
NP 000873.2 (human), AAD56385.1 (human), NP 001152896.1 (mouse), and
NP 032377.1 (mouse).
[0144] It is understood that the specific cleavage site of a signal
peptide may vary by 1, 2,
3 or more residues. Accordingly, in certain embodiments, mature p35
polypeptides of the
invention include the predicted mature sequence consisting of residues 57 to
253 of SEQ
ID NO: 4 as well as mature sequences consisting of amino acids 52 to 253, 53
to 253, 54
to 253, 55 to 253, 56 to 253, 58 to 253, 59 to 253, 60 to 253, 61 to 263 and
62 to 253 of
SEQ ID NO: 4.
[0145]
Suitable IL-12 p35 domains may be truncated at the C-terminus by one or more
amino acid residues.
Therefore, in additional embodiments the IL-12 p35 dom amn
comprises, or alternatively consists of, a fragment of SEQ ID NO: 4 beginning
with
residue 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 of SEQ ID NO: 4 and
ending with
residue 247, 248, 249, 250, 251, 252, or 253 of SEQ ID NO: 4.
[0146] The optional second peptide linker (iv) may be any suitable
peptide linker that
allows folding of the scIL-12 polypeptide into a functional protein. In
certain
embodiments, the optional second peptide linker consists of 10 or fewer amino
acids. In
specific embodiments, the second peptide linker consists of 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10
amino acids. In specific embodiments, the second peptide linker comprises any
sequence
and combination of one or more amino acids selected from: Glycine (Gly);
Serine (Ser);
Alanine (Ala); Threonine (Thr); and, Proline (Pro). In preferred embodiments,
the second
peptide linker is selected from the peptides Thr-Pro-Ser (SEQ ID NO: 41) and
Ser-Gly-
Pro-Ala-Pro (SEQ ID NO: 42), and peptides with one amino acid substitution in
Thr-Pro-
Ser (SEQ ID NO: 41) and Ser-Gly-Pro-Ala-Pro (SEQ ID NO: 42). In certain
embodiments the second peptide linker is absent. In a preferred embodiment,
the first and
second peptide linkers consist of 10 or fewer amino acid residues combined.
[0147] In certain embodiments, the second IL-12 p40 domain (p40C) is a
C-terminal
fragment of an IL-12 p40 subunit. C-terminal fragments of p40 suitable as a
second IL-
12 p40 domain (p40C) comprise, or alternatively consist of, amino acids 289 to
328, 290

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to 329, 291 to 328, 292 to 328, 293 to 328, 294 to 328, 295 to 328, 296 to
328, 297 to
328, 298 to 328, and 299 to 328 of SEQ ID NO: 2.
[0148] Suitable second IL-12 p40 domains (p40C) may be truncated at the C-
terminus by
one or more amino acid residues. Therefore, in additional embodiments the
second IL-12
p40 domain (p40C) comprises, or alternatively consists of, a fragment of SEQ
ID NO: 2
beginning with residue 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, or
299 of SEQ
ID NO: 2 and ending with residue 322, 323, 324, 325, 326, 327, or 328 of SEQ
ID NO: 2.
[0149] The full-length sequence of a representative scIL-12 polypeptide of
the invention
is presented herein as SEQ ID NO: 10. The full-length sequence contains a
predicted
signal peptide at amino acids 1 to 22 of SEQ ID NO: 10, and a mature scIL-12
polypeptide at amino acids 23 to 533 of SEQ ID NO: 10.
[0150] In another specific embodiment, the scIL-12 polypeptide is encoded
by a
polynucleotide comprising a nucleic acid sequence selected from the group
consisting of
SEQ ID NO: 9 and nucleotides 67 to 1599 of SEQ ID NO: 9.
[0151] Thus, a first subject of the invention relates to an isolated scIL-
12 polypeptide. In
a specific embodiment, the isolated polypeptide comprises an amino acid
sequence
selected from the group consisting of SEQ ID NO: 10 and amino acids 23 to 533
of SEQ
ID NO: 10.
[0152] One of skill in the art is able to produce other polynucleotides to
encode the
polypeptides of the invention, by making use of the present invention and the
degeneracy
or non-universality of the genetic code as described herein.
[0153] Additional embodiments of the present invention include functional
fragments of
a scIL-12 polypeptide, or fusion proteins comprising a scIL-12 polypeptide of
the present
invention fused to second polypeptide comprising a heterologous, or normally
non-
contiguous, protein domain. Preferably, the second polypeptide is a targeting
polypeptide
such as an antibody, including single chain antibodies or antibody fragments.
Thus, the
invention provides a ScIL-12 polypeptide fused at its N- or C-terminus to a
second
polypeptide, preferably to an antibody, an antibody fragment, or a single
chain antibody.
[0154] The invention also provides variants of the scIL-12 polypeptides of
the invention.
In certain embodiments a scIL-12 variant polypeptide is at least 80%, at least
85%, at
least 90%, at or at least 95%, at least 97%, at least 98%, or at least 99%
identical to the
full-length or mature amino acid sequence of SEQ ID NO: 10, where the variant

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polypeptide exhibits at least one IL-12 activity, such as induction of IFN-
gamma
secretion from NK cells. Such IL-12 activities are readily determined using
assays
known in the art, such as the assays described in Example 8 of US Patent
5,457,038,
which is incorporated herein by reference.
[0155] The present invention also relates to compositions comprising an
isolated
polypeptide according to the invention.
Compositions
[0156] The present invention also relates to compositions comprising the
scIL-12
polynucleotides or polypeptides according to the invention. S uch compositions
may
comprise a scIL-12 polypeptide or a polynucleotide encoding a scIL-12
polypeptide, as
defined above, and an acceptable carrier or vehicle. The compositions of the
invention
are particularly suitable for formulation of biological material for use in
therapeutic
administration. Thus, in one embodiment, the composition comprises a
polynucleotide
encoding a scIL-12 polypeptide. In another embodiment, the composition
comprises a
scIL-12 polypeptide according to the invention.
[0157] The phrase "acceptable" refers to molecular entities and
compositions that are
physiologically tolerable to the cell or organism when administered. The term
"carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
composition is
administered. Such carriers can be sterile liquids, such as water and oils,
including those
of petroleum, animal, vegetable or synthetic origin, such as peanut oil,
soybean oil,
mineral oil, sesame oil and the like. Examples of acceptable carriers are
saline, buffered
saline, isotonic saline (e.g., monosodium or disodium phosphate, sodium,
potassium,
calcium or magnesium chloride, or mixtures of such salts), Ringer's solution,
dextrose,
water, sterile water, glycerol, ethanol, and combinations thereof 1,3 -
butanediol and
sterile fixed oils are conveniently employed as solvents or suspending media.
Any bland
fixed oil can be employed including synthetic mono- or di-glycerides. Fatty
acids such as
oleic acid also find use in the preparation of injectables. Water or aqueous
solution saline
solutions and aqueous dextrose and glycerol solutions are preferably employed
as
carriers, particularly for injectable solutions. S uitable pharmaceutical
carriers are
described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
Pharmaceutical
compositions of the invention may be formulated for the purpose of topical,
oral,

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parenteral, intranasal, intravenous, intramuscular, intratumoral,
subcutaneous, intraocular,
and the like, administration.
[0158] Preferably, the compositions comprise an acceptable vehicle for an
injectable
formulation. This vehicle can be, in particular, a sterile, isotonic saline
solution
(monosodium or disodium phosphate, sodium, potassium, calcium or magnesium
chloride, and the like, or mixtures of such salts), or dry, in particular
lyophilized,
compositions which, on addition, as appropriate, of sterilized water or of
physiological
saline, enable injectable solutions to be formed. T he preferred sterile
injectable
preparations can be a solution or suspension in a nontoxic parenterally
acceptable solvent
or diluent.
[0159] In yet another embodiment, a composition comprising a scIL-12
polypeptide, or
polynucleotide encoding the polypeptide, can be delivered in a controlled
release system.
For example, the polynucleotide or polypeptide may be administered using
intravenous
infusion, an implantable osmotic pump, a transdermal patch, liposomes, or
other modes of
administration. Other controlled release systems are discussed in the review
by Langer
[Science 249:1527-1533 (1990)].
Expression of Single Chain IL-12 Polypeptides
[0160] With the sequence of the scIL-12 polypeptides and the
polynucleotides encoding
them, large quantities of scIL-12 polypeptides may be prepared. By the
appropriate
expression of vectors in cells, high efficiency production may be achieved.
Thereafter,
standard purification methods may be used, such as ammonium sulfate
precipitations,
column chromatography, electrophoresis, centrifugation, crystallization and
others. See
various volumes of Methods in Enzymology for techniques typically used for
protein
purification. Alternatively, in some embodiments high efficiency of production
is
unnecessary, but the presence of a known inducing protein within a carefully
engineered
expression system is quite valuable. Typically, the expression system will be
a cell, but an
in vitro expression system may also be constructed.
[0161] A polynucleotide encoding a scIL-12, or fragment, derivative or
analog thereof, or
a functionally active derivative, including a chimeric protein, thereof, can
be inserted into
an appropriate expression vector, i.e., a vector which comprises the necessary
elements
for the transcription and translation of the inserted protein-coding sequence.
A
polynucleotide of the invention is operationally linked with a transcriptional
control

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sequence in an expression vector. A n expression vector also preferably
includes a
replication origin.
[0162] The isolated polynucleotides of the invention may be inserted into
any appropriate
cloning vector. A large number of vector-host systems known in the art may be
used.
Possible vectors include, but are not limited to, plasmids or modified
viruses, but the
vector system must be compatible with the host cell used. Examples of vectors
include,
but are not limited to, Escherichia coli, bacteriophages such as lambda
derivatives, or
plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX
vectors,
pmal-c, pFLAG, etc. The insertion into a cloning vector can, for example, be
accomplished by ligating the polynucleotide into a cloning vector that has
complementary
cohesive termini. However, if the complementary restriction sites used to
fragment the
polynucleotide are not present in the cloning vector, the ends of the
polynucleotide
molecules may be enzymatically modified. A lternatively, any site desired may
be
produced by ligating nucleotide sequences (linkers) onto the DNA termini;
these ligated
linkers may comprise specific chemically synthesized oligonucleotides encoding
restriction endonuclease recognition sequences. Preferably, the cloned gene is
contained
on a shuttle vector plasmid, which provides for expansion in a cloning cell,
e.g., E. coli,
and purification for subsequent insertion into an appropriate expression cell
line, if such is
desired. For example, a shuttle vector, which is a vector that can replicate
in more than
one type of organism, can be prepared for replication in both E. coli and
Saccharomyces
cerevisiae by linking sequences from an E. coli plasmid with sequences form
the yeast 2i,t
plasmid.
[0163] In addition, the present invention relates to an expression vector
comprising a
polynucleotide according the invention, operatively linked to a transcription
regulatory
element. In one embodiment, the polynucleotide is operatively linked with an
expression
control sequence permitting expression of the scIL-12 polypeptide in an
expression
competent host cell. The expression control sequence may comprise a promoter
that is
functional in the host cell in which expression is desired. The vector may be
a plasmid
DNA molecule or a viral vector. In certain embodiments, viral vectors include,
without
limitation, retrovirus, adenovirus, adeno-associated virus (AAV), herpes
virus, and
vaccinia virus. The invention further relates to a replication defective
recombinant virus
comprising in its genome, a polynucleotide according to the invention. Thus,
the present

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invention also relates to an isolated host cell comprising such an expression
vector,
wherein the transcription regulatory element is operative in the host cell.
[0164] The desired genes will be inserted into any of a wide selection of
expression
vectors. The selection of an appropriate vector and cell line depends upon the
constraints
of the desired product. Typical expression vectors are described in Sambrook
et al.
(1989). Suitable cell lines may be selected from a depository, such as the
ATCC. See,
ATCC Catalogue of Cell Lines and Hybridomas (6th ed.) (1988); ATCC Cell Lines,
Viruses, and Antisera, each of which is hereby incorporated herein by
reference. The
vectors are introduced to the desired cells by standard transformation or
transfection
procedures as described, for instance, in Sambrook et al. (1989).
Fusion proteins will typically be made by either recombinant nucleic acid
methods or by
synthetic polypeptide methods. Techniques for nucleic acid manipulation are
described
generally, for example, in Sambrook et al. (1989), Molecular Cloning: A
Laboratory
Manual (2d ed.), Vols. 1-3, Cold Spring Harbor Laboratory, which are
incorporated
herein by reference. Techniques for synthesis of polypeptides are described,
for example,
in Merrifield, J. Amer. Chem. Soc. 85:2149-2156 (1963).
[0165] Once a particular recombinant DNA molecule is identified and
isolated, any of
multiple methods known in the art may be used to propagate it. Once a suitable
host
system and growth conditions are established, recombinant expression vectors
can be
propagated and prepared in quantity. As previously explained, the expression
vectors
which can be used include, but are not limited to, the following vectors or
their
derivatives: hum an or animal viruses such as vaccinia virus, adenovirus, or
adeno-
associated virus (AAV); insect viruses such as baculovirus; yeast vectors;
bacteriophage
vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.
[0166] In addition, a host cell strain may be chosen which modulates the
expression of
the inserted sequences, or modifies and processes the gene product in the
specific fashion
desired. D ifferent host cells have characteristic and specific mechanisms for
the
translational and post-translational processing and modification of proteins.
Appropriate
cell lines or host systems can be chosen to ensure the desired modification
and processing
of the foreign protein expressed. Expression in yeast can produce a
biologically active
product. Expression in eukaryotic cells can increase the likelihood of
"native" folding.
Moreover, expression in mammalian cells can provide a tool for reconstituting,
or

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constituting, scIL-12 activity. Furthermore, different vector/host expression
systems may
affect processing reactions, such as proteolytic cleavages, to a different
extent.
[0167] Vectors are introduced into the desired host cells by methods known
in the art,
e.g., transfection, electroporation, microinjection, transduction, cell
fusion, DEAE
dextran, calcium phosphate precipitation, lipofection (lysosome fusion),
particle
bombardment, use of a gene gun, or a DNA vector transporter (see, e.g., Wu et
al., 1992,
J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624;
Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15,
1990).
[0168] Soluble forms of the protein can be obtained by collecting culture
fluid, or
solubilizing inclusion bodies, e.g., by treatment with detergent, and if
desired sonication
or other mechanical processes, as described above. The solubilized or soluble
protein can
be isolated using various techniques, such as polyacrylamide gel
electrophoresis (PAGE),
isoelectric focusing, 2-dimensional gel electrophoresis, chromatography (e.g.,
ion
exchange, affinity, immunoaffinity, and sizing column chromatography),
centrifugation,
differential solubility, immunoprecipitation, or by any other standard
technique for the
purification of proteins.
Vectors and Gene Expression Cassettes Comprising scIL-12 Polynucleotides
[0169] The present invention also relates to a vector comprising a
polynucleotide
encoding a scIL-12 polypeptide according to the invention. The present
invention also
provides a gene expression cassette comprising a polynucleotide encoding a
scIL-12
polypeptide according to the invention. The polynucleotides of the invention,
where
appropriate incorporated in vectors or gene expression cassettes, and the
compositions
comprising them, are useful for enhancing immune system function, for example
as
vaccine adjuvants and in combination with other immunomodulators and/or small
molecule pharmaceuticals in the treatment of infections and cancer. They may
be used for
the transfer and expression of genes in vitro or in vivo in any type of cell
or tissue. The
transformation can, moreover, be targeted (transfer to a particular tissue
can, in particular,
be determined by the choice of a vector, and expression by the choice of a
particular
promoter). The polynucleotides and vectors of the invention are advantageously
used for
the production in vivo of scIL-12 polypeptides of the invention.
[0170] The polynucleotides encoding the scIL-12 polypeptides of the
invention may be
used in a plasmid vector. Preferably, an expression control sequence is
operably linked to

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the scIL-12 polynucleotide coding sequence for expression of the scIL-12
polypeptide.
The expression control sequence may be any enhancer, response element, or
promoter
system in vectors capable of transforming or transfecting a host cell. Once
the vector has
been incorporated into the appropriate host, the host, depending on t he use,
will be
maintained under conditions suitable for high level expression of the
polynucleotides.
[0171] Polynucleotides will normally be expressed in hosts after the
sequences have been
operably linked to (i.e., positioned to ensure the functioning of) an
expression control
sequence. These expression vectors are typically replicable in the host
organisms either as
episomes or as an integral part of the host chromosomal DNA. Commonly,
expression
vectors will contain selection markers, e.g., tetracycline or neomycin, to
permit detection
of those cells transformed with the desired DNA sequences (see, e.g., U.S.
Pat. No.
4,704,362, which is incorporated herein by reference).
[0172] Escherichia coli is one prokaryotic host useful for cloning the
polynucleotides of
the present invention. Other microbial hosts suitable for use include, without
limitation,
bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as
Salmonella,
Serratia, and various Pseudomonas species.
[0173] Other eukaryotic cells may be used, including, without limitation,
yeast cells,
insect tissue culture cells, avian cells or the like. Preferably, mammalian
tissue cell
culture will be used to produce the polypeptides of the present invention
(see, Winnacker,
From Genes to Clones, VCH Publishers, N.Y. (1987), which is incorporated
herein by
reference).
[0174] Expression vectors may also include, without limitation, expression
control
sequences, such as an origin of replication, a promoter, an enhancer, a
response element,
and necessary processing information sites, such as ribosome-binding sites,
RNA splice
sites, polyadenylation sites, and transcriptional terminator sequences.
Preferably, the
enhancers or promoters will be those naturally associated with genes encoding
the IL-12
subunits p40 and p35, although it will be understood that in many cases others
will be
equally or more appropriate. In further embodiments, expression control
sequences are
enhancers or promoters derived from viruses, such as 5V40, Adenovirus, Bovine
Papilloma Virus, and the like.
[0175] The vectors comprising the polynucleotides of the present invention
can be
transferred into the host cell by well-known methods, which vary depending on
the type

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of cellular host. For example, calcium chloride transfection is commonly
utilized for
procaryotic cells, whereas calcium phosphate treatment may be used for other
cellular
hosts. (See, generally, Sambrook et al. (1989), Molecular Cloning: A
Laboratory Manual
(2d ed.), Cold Spring Harbor Press, which is incorporated herein by
reference.) The term
"transformed cell" is meant to also include the progeny of a transformed cell.
[0176] Potential host-vector systems include but are not limited to
mammalian cell
systems infected with virus (e.g., vaccinia virus, adenovirus, adeno-
associated virus, etc.);
insect cell systems infected with virus (e.g., baculovirus); microorganisms
such as yeast
containing yeast vectors; or bacteria transformed with bacteriophage, DNA,
plasmid
DNA, or cosmid DNA. The expression elements of vectors vary in their strengths
and
specificities. Depending on the host-vector system utilized, any one of a
number of
suitable transcription and translation elements may be used.
[0177] A recombinant scIL-12 protein of the invention, or functional
fragment,
derivative, chimeric construct, or analog thereof, may be expressed
chromosomally, after
integration of the coding sequence by recombination. In this regard, any of a
number of
amplification systems may be used to achieve high levels of stable gene
expression (See
Sambrook et al., 1989, supra).
[0178] The cell containing the recombinant vector comprising the scIL-12
polynucleotide
is cultured in an appropriate cell culture medium under conditions that
provide for
expression of the scIL-12 polypeptide by the cell. A ny of the methods
previously
described for the insertion of DNA fragments into a cloning vector may be used
to
construct expression vectors containing a gene consisting of appropriate
transcriptional/translational control signals and the protein coding
sequences. T hese
methods may include in vitro recombinant DNA and synthetic techniques and in
vivo
recombination (genetic recombination).
[0179] A polynucleotide encoding a scIL-12 polypeptide may be operably
linked and
controlled by any regulatory region, i.e., promoter/enhancer element known in
the art, but
these regulatory elements must be functional in the host cell selected for
expression. The
regulatory regions may comprise a promoter region for functional transcription
in the host
cell, as well as a region situated 3' of the gene of interest, and which
specifies a signal for
termination of transcription and a polyadenylation site. All these elements
constitute an
expression cassette.

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[0180] Expression vectors comprising a polynucleotide encoding a scIL-12
polypeptide
of the invention can be identified by five general approaches: (a) PCR
amplification of
the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c)
presence
or absence of selection marker gene functions, (d) analyses with appropriate
restriction
endonucleases, and (e) expression of inserted sequences. In the first
approach, the nucleic
acids can be amplified by PCR to provide for detection of the amplified
product. In the
second approach, the presence of a foreign gene inserted in an expression
vector can be
detected by nucleic acid hybridization using probes comprising sequences that
are
homologous to an inserted marker gene. In the third approach, the recombinant
vector/host system can be identified and selected based upon the presence or
absence of
certain "selection marker" gene functions (e.g., I3-galactosidase activity,
thymidine kinase
activity, resistance to antibiotics, transformation phenotype, occlusion body
formation in
baculovirus, etc.) caused by the insertion of foreign genes in the vector. In
another
example, if the nucleic acid encoding a scIL-12 polypeptide is inserted within
the
"selection marker" gene sequence of the vector, recombinants comprising the
scIL-12
nucleic acid insert can be identified by the absence of the gene function. In
the fourth
approach, recombinant expression vectors are identified by digestion with
appropriate
restriction enzymes. In the fifth approach, recombinant expression vectors can
be
identified by assaying for the activity, biochemical, or immunological
characteristics of
the gene product expressed by the recombinant, provided that the expressed
protein
assumes a functionally active conformation.
[0181] A wide variety of host/expression vector combinations may be
employed in
expressing the DNA sequences of this invention. Useful expression vectors, for
example,
may consist of segments of chromosomal, non-chromosomal and synthetic DNA
sequences. Suitable vectors include but are not limited to derivatives of 5V40
and known
bacterial plasmids, e.g., E. coli plasmids col El, pCR1, pBR322, pMal-C2, pET,
pGEX
(Smith et at., 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such
as RP4;
phage DNAS, e.g., the numerous derivatives of phage 1, e.g., NM989, and other
phage
DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such
as the
2m plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as
vectors
useful in insect or mammalian cells; vectors derived from combinations of
plasmids and

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phage DNAs, such as plasmids that have been modified to employ phage DNA or
other
expression control sequences; and the like.
[0182] The present invention also provides a gene expression cassette
that is capable of
being expressed in a h ost cell, wherein the gene expression cassette
comprises a
polynucleotide that encodes a scIL-12 polypeptide according to the invention.
T hus,
Applicants' invention also provides novel gene expression cassettes useful in
a scIL-12
expression system.
[0183] Gene expression cassettes of the invention may include a gene
switch to allow the
regulation of gene expression by addition or removal of a specific ligand. In
one
embodiment, the gene switch is one in which the level of gene expression is
dependent on
the level of ligand that is present. Examples of ligand-dependent
transcription factor
complexes that may be used in the gene switches of the invention include,
without
limitation, members of the nuclear receptor superfamily activated by their
respective
ligands glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs
and mimetics
thereof); rTTA activated by tetracycline; Biotin-based switch systems;
FKBP/rapamycin
switch systems; cumate switch systems; riboswitch systems; among others.
[0184] In one aspect of the invention, the gene switch is an EcR-based
gene switch.
Examples of such systems include, without limitation, the systems described
in:
PCT/US2001/009050 (WO 2001/070816); U .S. Pat. Nos. 7,091,038; 7,776,587;
7,807,417; 8,202,718; P CT/US2001/030608 (WO 2002/029075); U.S. Pat. Nos.
8,105,825; 8,168,426; P CT/U52002/005235 (WO 2002/066613); U.S. App. No.
10/468,200 (U.S. Pub. No. 20120167239); P CT/U52002/005706 (WO 2002/066614);
U.S. Pat. Nos. 7,531,326; 8,236,556; 8, 598,409;
PCT/U52002/005090 (WO
2002/066612); U .S. App. No. 10/468,193 (U.S. Pub. No. 20060100416);
PCT/U52002/005234 (WO 2003/027266); U .S. Pat. Nos. 7,601,508; 7,829,676;
7,919,269; 8,030,067; P CT/U52002/005708 (WO 2002/066615); U.S. App. No.
10/468,192 (U.S. Pub. No. 20110212528); P CT/US2002/005026 (WO 2003/027289);
U.S. Pat. Nos. 7,563,879; 8,021,878; 8, 497,093;
PCT/U52005/015089 (WO
2005/108617); U .S. Pat. No. 7,935,510; 8, 076,454; P CT/U52008/011270 (WO
2009/045370); U .S. App. No. 12/241,018 (U.S. Pub. No. 20090136465);
PCT/U52008/011563 (WO 2009/048560); U.S. App. No. 12/247,738 (U.S. Pub. No.
20090123441); P CT/US2009/005510 (WO 2010/042189); U.S. App. No. 13/123,129

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(U.S. Pub. No. 20110268766); PCT/US2011/029682 (WO 2011/119773); U.S. App. No.
13/636,473 (U.S. Pub. No. 20130195800); P CT/US2012/027515 (WO 2012/122025);
and, U.S. App. No. 14/001,943 (U.S. Pub. No. [Pending]), each of which is
incorporated
by reference in its entirety.
[0185] In another aspect of the invention, the gene switch is based on
heterodimerization
of FK506 binding protein (FKBP) with FKBP rapamycin associated protein (FRAP)
and
is regulated through rapamycin or its non-immunosuppressive analogs. Examples
of such
systems include, without limitation, the ARGENTTm Transcriptional Technology
(ARIAD Pharmaceuticals, Cambridge, Mass.) and the systems described in U.S.
Pat. Nos.
6,015,709, 6,117,680, 6,479,653, 6,187,757, and 6,649,595.
[0186] In another aspect of the invention, gene expression cassettes of
the invention
incorporate a cumate switch system, which works through the CymR repressor
that binds
the cumate operator sequences with high affinity. (SparQTM Cumate Switch,
System
Biosciences, Inc.) T he repression is alleviated through the addition of
cumate, a non-
toxic small molecule that binds to CymR. This system has a dynamic
inducibility, can be
finely tuned and is reversible and inducible.
[0187] In another aspect of the invention, gene expression cassettes of
the invention
incorporate a riboswitch, which is a regulatory segment of a messenger RNA
molecule
that binds an effector, resulting in a change in production of the proteins
encoded by the
mRNA. An mRNA that contains a riboswitch is directly involved in regulating
its own
activity in response to the concentrations of its effector molecule. E
ffectors can be
metabolites derived from purine/pyrimidine, amino acid, vitamin, or other
small molecule
co-factors. These effectors act as ligands for the riboswitch sensor, or
aptamer. Breaker,
RR. Mol Cell. (2011) 43(6):867-79.
[0188] In another aspect of the invention, gene expression cassettes of
the invention
incorporate the biotin-based gene switch system, in which the bacterial
repressor protein
TetR is fused to streptavidin, which interacts with the synthetic
biotinylation signal
AVITAG that is fused to VP16 to activate gene expression. Biotinylation of the
AVITAG peptide is regulated by a bacterial biotin ligase BirA, thus enabling
ligand
responsiveness. Weber et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 2643-
2648;
Weber et al. (2009) Metabolic Engineering, 11(2):117-124.

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[0189]
Additional gene switch systems appropriate for use in the instant invention
are
well known in the art, including but not limited to those described in
Auslander and
Fussenegger, Trends in Biotechnology (2012), 31(3):155-168, incorporated
herein by
reference.
[0190] Examples of ligands for use in gene switch systems include,
without limitation, an
ecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone
A, and
the like, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-
diacylhydrazines such
as those disclosed in U.S. Pat. Nos. 6,013,836; 5,117,057; 5,530,028; and
5,378,726 and
U.S. Published Application Nos. 2005/0209283 and 2006/0020146; oxadiazolines
as
described in U.S. Published Application No. 2004/0171651; dibenzoylalkyl
cyanohydrazines such as those disclosed in European Application No. 461,809; N-
alkyl-
N,N'-diaroylhydrazines such as those disclosed in U.S. Pat. No. 5,225,443; N-
acyl-N-
alkylcarbonylhydrazines such as those disclosed in European Application No.
234,994;
N-aroyl-N-alkyl-N'-aroylhydrazines such as those described in U.S. Pat. No.
4,985,461;
arnidoketones such as those described in U.S. Published Application No.
2004/0049037;
each of which is incorporated herein by reference and other similar materials
including
3,5 -di-tert-butyl-4-hydroxy-N-isobutyl-b enzamide,
8-0-acetylharpagide, oxysterols,
22(R) hydroxycholesterol, 24(5) hydroxycholesterol, 25-epoxycholesterol,
T0901317, 5-
alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate,
framesol,
bile acids, 1,1-biphosphonate esters, juvenile hormone III, and the like.
Examples of
diacylhydrazine ligands useful in the present invention include RG-115819 (3,5-
Dimethyl-b enzoic acid
N-(1 -ethyl-2,2-dimethyl-propy1)-N'-(2-methyl-3 -methoxy-
b enzoy1)-hydrazide- ), RG-115932 ((R)-3,5-Dimethyl-benzoic acid N-(1 -tert-
butyl-
buty1)-N'-(2-ethy1-3 -methoxy-b enzoy1)-hydrazide), and RG-115830 (3,5 -
Dimethyl-
b enzoic acid N-(1 -tert-butyl-butyl)-N' -(2- ethy1-3 -methoxy-b enzoy1)-
hydrazide). See, e.g.,
U.S. patent application Ser. No. 12/155,111, and PCT Appl. No.
PCT/U52008/006757,
both of which are incorporated herein by reference in their entireties.
Antibodies to Single Chain IL-12 Polypeptides
[0191]
According to the invention, a scIL-12 polypeptide produced recombinantly or by
chemical synthesis, and fragments or other derivatives or analogs thereof,
including
fusion proteins, may be used as an antigen or immunogen to generate
antibodies.
Preferably, the antibodies specifically bind scIL-12 polypeptides, but do not
bind native

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IL-12 polypeptides. M ore preferably, the antibodies specifically bind a scIL-
12
polypeptide, but do not bind other cytokine polypeptides.
[0192] In another embodiment, the invention relates to an antibody which
specifically
binds an antigenic peptide comprising a fragment of a scIL-12 polypeptide
according to
the invention as described above. The antibody may be polyclonal or monoclonal
and
may be produced by in vitro or in vivo techniques.
[0193] The antibodies of the invention possess specificity for binding to
particular scIL-
12 polypeptides. Thus, reagents for determining qualitative or quantitative
presence of
these or homologous polypeptides may be produced. Alternatively, these
antibodies may
be used to separate or purify scIL-12 polypeptides.
[0194] For production of polyclonal antibodies, an appropriate target
immune system is
selected, typically a mouse or rabbit. The substantially purified antigen is
presented to the
immune system in a fashion determined by methods appropriate for the animal
and other
parameters well known to immunologists. Typical sites for injection are in the
footpads,
intramuscularly, intraperitoneally, or intradermally. Of course, another
species may be
substituted for a mouse or rabbit.
[0195] An immunological response is usually assayed with an immunoassay.
Normally
such immunoassays involve some purification of a source of antigen, for
example,
produced by the same cells and in the same fashion as the antigen was
produced. The
immunoassay may be a radioimmunoassay, an enzyme-linked assay (ELISA), a
fluorescent assay, or any of many other choices, most of which are
functionally
equivalent but may exhibit advantages under specific conditions.
[0196] Monoclonal antibodies with high affinities are typically made by
standard
procedures as described, e.g., in Harlow and Lane (1988), Antibodies: A
Laboratory
Manual, Cold Spring Harbor Laboratory; or Goding (1986), Monoclonal
Antibodies:
Principles and Practice (2d ed) Academic Press, New York, which are hereby
incorporated herein by reference. Briefly, appropriate animals will be
selected and the
desired immunization protocol followed. After the appropriate period of time,
the spleens
of such animals are excised and individual spleen cells fused, typically, to
immortalized
myeloma cells under appropriate selection conditions. Thereafter, the cells
are clonally
separated and the supernatants of each clone are tested for their production
of an
appropriate antibody specific for the desired region of the antigen.

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[0197] Other suitable techniques involve in vitro exposure of lymphocytes
to the
antigenic polypeptides or alternatively to selection of libraries of
antibodies in phage or
similar vectors. See, Huse et al., (1989) "Generation of a Large Combinatorial
Library of
the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281, hereby
incorporated herein by reference.
[0198] The polypeptides and antibodies of the present invention may be
used with or
without modification. Frequently, the polypeptides and antibodies will be
labeled by
joining, either covalently or non-covalently, a substance which provides for a
detectable
signal. A wide variety of labels and conjugation techniques are known and are
reported
extensively in both the scientific and patent literature. Suitable labels
include, without
limitation, radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescence,
chemiluminescence, magnetic particles and the like. Patents, teaching the use
of such
labels include US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437;
4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced,
see
Cabilly, US Patent 4,816,567.
[0199] A molecule is "antigenic" when it is capable of specifically
interacting with an
antigen recognition molecule of the immune system, such as an immunoglobulin
(antibody) or T cell antigen receptor. An antigenic polypeptide contains at
least about 5,
and preferably at least about 10 amino acids. An antigenic portion of a
molecule can be
that portion that is immunodominant for antibody or T cell receptor
recognition, or it can
be a portion used to generate an antibody to the molecule by conjugating the
antigenic
portion to a carrier molecule for immunization. A molecule that is antigenic
need not be
itself immunogenic, i.e., capable of eliciting an immune response without a
carrier.
[0200] Such antibodies include but are not limited to polyclonal,
monoclonal, chimeric,
single chain, Fab fragments, and an Fab expression library. The scIL-12
antibodies of the
invention may be cross reactive, e.g., they may recognize scIL-12 polypeptides
derived
from different species. Polyclonal antibodies have greater likelihood of cross
reactivity.
Alternatively, an antibody of the invention may be specific for a single form
of scIL-12
polyptide, such as a human scIL-12 polypeptide. Preferably, such an antibody
is specific
for human scIL-12.
[0201] Various procedures known in the art may be used for the production
of polyclonal
antibodies. For the production of antibody, various host animals can be
immunized by

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injection with a scIL-12 polypeptide, or a derivative (e.g., fragment or
fusion protein)
thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
In one
embodiment, the scIL-12 polypeptide or fragment thereof can be conjugated to
an
immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet
hemocyanin
(KLH). Various adjuvants may be used to increase the immunological response,
depending on t he host species, including but not limited to Freund's
(complete and
incomplete), mineral gels such as aluminum hydroxide, surface active
substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum.
[0202] For preparation of monoclonal antibodies directed toward a scIL-12
polypeptide,
or fragment, analog, or derivative thereof, any technique that provides for
the production
of antibody molecules by continuous cell lines in culture may be used. These
include but
are not limited to the hybridoma technique originally developed by Kohler and
Milstein
[Nature 256:495-497 (1975)], as well as the trioma technique, the human B-cell
hybridoma technique [Kozbor et al., Immunology Today 4:72 1983); Cote et al.,
Proc.
Natl. Acad. Sci. U.S.A. 80:2026-2030 (1983)], and the EBV-hybridoma technique
to
produce human monoclonal antibodies [Cole et al., in Monoclonal Antibodies and
Cancer
Therapy, Alan R. Liss, Inc., pp. 77 -96 (1985)]. In an additional embodiment
of the
invention, monoclonal antibodies can be produced in germ-free animals
[International
Patent Publication No. WO 89/12690, published 28 December 1989]. In fact,
according
to the invention, techniques developed for the production of "chimeric
antibodies"
[Morrison et al., J. Bacteriol. 159:870 (1984); Neuberger et al., Nature
312:604-608
(1984); Takeda et al., Nature 314:452-454 (1985)] by splicing the genes from a
mouse
antibody molecule specific for a scIL-12 polypeptide together with genes from
a human
antibody molecule of appropriate biological activity can be used; such
antibodies are
within the scope of this invention. S uch human or humanized chimeric
antibodies are
preferred for use in therapy of human diseases or disorders (described infra),
since the
human or humanized antibodies are much less likely than xenogenic antibodies
to induce
an immune response, in particular an allergic response, themselves.
[0203] According to the invention, techniques described for the production
of single
chain Fv (scFv) antibodies [U.S. Patent Nos. 5,476,786 and 5,132,405 to
Huston; U.S.

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Patent 4,946,778] can be adapted to produce scIL-12 polypeptide-specific
single chain
antibodies. An additional embodiment of the invention utilizes the techniques
described
for the construction of Fab expression libraries [Huse et al., Science
246:1275-1281
(1989)] to allow rapid and easy identification of monoclonal Fab fragments
with the
desired specificity for a scIL-12 polypeptide, or its derivatives, or analogs.
[0204] Antibody fragments which contain the idiotype of the antibody
molecule can be
generated by known techniques. For example, such fragments include but are not
limited
to: the F(ab')2 fragment which can be produced by pepsin digestion of the
antibody
molecule; the Fab' fragments which can be generated by reducing the disulfide
bridges of
the F(ab')2 fragment, and the Fab fragments which can be generated by treating
the
antibody molecule with papain and a reducing agent.
[0205] In the production of antibodies, screening for the desired antibody
can be
accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA
(enzyme-
linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric
assays, gel
diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays
(using
colloidal gold, enzyme or radioisotope labels, for example), western blots,
precipitation
reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination assays),
complement fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody binding is
detected by
detecting a label on the primary antibody. In another embodiment, the primary
antibody
is detected by detecting binding of a secondary antibody or reagent to the
primary
antibody. In a further embodiment, the secondary antibody is labeled. Many
means are
known in the art for detecting binding in an immunoassay and are within the
scope of the
present invention. For example, to select antibodies which recognize a
specific epitope of
a scIL-12 polypeptide, one may assay generated hybridomas for a product which
binds to
a scIL-12 polypeptide fragment containing such epitope.
[0206] The foregoing antibodies can be used in methods known in the art
relating to the
localization and activity of a scIL-12 polypeptide, e.g., for western
blotting, imaging a
scIL-12 polypeptide in situ, measuring levels thereof in appropriate
physiological
samples, etc. using any of the detection techniques mentioned above or known
in the art.

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USES OF SINGLE CHAIN IL-12 POLYNUCLEOTIDES AND POLYPEPTIDES
[0207] The scIL-12 polypeptides and polynucleotides of the present
invention have a
variety of utilities. For example, the polynucleotides and polypeptides of the
invention are
useful in the treatment of diseases in which stimulation of immune function
might be
beneficial. In specific embodiments, the scIL-12 polypeptides and
polynucleotides of the
present invention are useful for the treatment of disease states responsive to
the enhanced
presence of gamma interferon; for the treatment of viral, bacterial, protozoan
and parasitic
infections; and for the treatment of proliferative disorders such as cancer.
The scIL-12
polynucleotides and polypeptides of the invention are also useful as vaccine
adjuvants.
Methods of Inducing IFN-gamma Production
[0208] The scIL-12 polypeptide and polynucleotide compositions of the
invention are
useful for inducing the production of IFN-gamma in a p atient in need thereof
Pathological states which benefit from IFN-gamma induction may result from
disease,
exposure to radiation or drugs, and include for example but without
limitation,
leukopenia, bacterial and viral infections, anemia, B cell or T cell
deficiencies including
immune cell or hematopoietic cell deficiency following a bone marrow
transplantation.
Methods of Treating Infections
[0209] The scIL-12 polypeptide and polynucleotide compositions according
to the
present invention can be used in the treatment of viral infections, including
without
limitation, HIV, Hepatitis A, Hepatitis B, Hepatitis C, rabies virus,
poliovirus, influenza
virus, meningitis virus, measles virus, mumps virus, rubella, pertussis,
encephalitis virus,
papilloma virus, yellow fever virus, respiratory syncytial virus, parvovirus,
chikungunya
virus, haemorrhagic fever viruses, Klebsiella, and Herpes viruses,
particularly, varicella,
cytomegalovirus and Epstein-Barr virus infection, among others.
[0210] The scIL-12 polypeptide and polynucleotide compositions according
to the
present invention can be used in the treatment of bacterial infections,
including, without
limitation, leprosy, tuberculosis, Yersinia pestis, Typhoid fever,
pneumococcal bacterial
infections, tetanus and anthrax, among others.
[0211] The scIL-12 polypeptide and polynucleotide compositions according
to the
present invention can also be used in the treatment of parasitic infections,
such as, but not

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limited to, leishmaniasis and malaria, among others; and protozoan infections,
such as,
but not limited to, T. cruzii) or helminths, such as Schistosoma.
Methods of Use as a Vaccine Adjuvant
[0212] The scIL-12 polypeptide and polynucleotide compositions are useful
as vaccine
adjuvants. B y "adjuvant" is meant a substance which enhances the immune
response
when administered together with an immunogen or antigen.
[0213] The scIL-12 polypeptide and polynucleotide compositions of the
invention are
useful for enhancing the immune response to viral vaccines, including without
limitation,
HIV, Hepatitis A, Hepatitis B, Hepatitis C, rabies virus, poliovirus,
influenza virus,
meningitis virus, measles virus, mumps virus, rubella, pertussis, encephalitis
virus,
papilloma virus, yellow fever virus, respiratory syncytial virus, parvovirus,
chikungunya
virus, haemorrhagic fever viruses, Klebsiella, and Herpes viruses,
particularly, varicella,
cytomegalovirus and Epstein-Barr virus.
[0214] The scIL-12 polypeptide and polynucleotide compositions of the
invention are
also useful for enhancing the immune response to bacterial vaccines, such as,
but not
limited to, vaccines against leprosy, tuberculosis, Yersinia pestis, Typhoid
fever,
pneumococcal bacteria, tetanus and anthrax, among others.
[0215] Similarly, polypeptides and polynucleotides of the invention are
also useful for
enhancing the immune response to vaccines against parasitic infections (such
as
leishmaniasis and malaria, among others) and vaccines against protozoan
infections (e.g.,
T. cruzii) or helminths, e.g., Schistosoma.
[0216] The scIL-12 polypeptide and polynucleotide compositions of the
invention are
also useful for enhancing the immune response to a therapeutic cancer vaccine.
A cancer
vaccine may comprise an antigen expressed on the surface of a cancer cell.
This antigen
may be naturally present on the cancer cell. Alternatively, the cancer cell
may be
manipulated ex vivo and transfected with a selected antigen, which it then
expresses when
introduced into the patient. A nonlimiting example of a cancer vaccine which
may be
enhanced by polynucleotides and polypeptides of the invention includes
Sipuleucel-T
(Provenge0).
[0217] Methods of formulating and administering vaccine adjuvants are
known in the art,
such as the methods described in US Patent 5,571,515, which are herein
incorporated by
reference.

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Methods of Treating Cancer
[0218] The scIL-12 polypeptide and polynucleotide compositions according
to the
present invention can be used to treat a cancer. Non-limiting examples of
cancers that can
be treated according to the invention include without limitation, breast
cancer, prostate
cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma,
malignant
melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck
cancer,
glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung
cancer, head or
neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-
cell lung
carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder
carcinoma,
pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma,
genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma,
multiple
myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma,
adrenal cortex
carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma,
choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical
hyperplasia,
leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute
myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic
leukemia,
acute granulocytic leukemia, hairy cell leukemia, neuroblastoma,
rhabdomyosarcoma,
Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's
disease, non-
Hodgkin's lymphoma, soft-tissue sarcoma, mesothelioma, osteogenic sarcoma,
primary
macroglobulinemia, and retinoblastoma, and the like.
[0219] The invention provides a method of treating cancer comprising
administering a
scIL-12 polyptide of the invention to a patient in a therapeutically effective
amount. In
certain embodiments the scIL-12 polypeptide is administered intratumorally.
[0220] The invention also provide a method of treating cancer comprising
administering
a scIL-12 polynucleotide of the invention to a patient in an amount sufficient
to produce a
therapeutically effective dose of scIL-12 polypeptide. In certain embodiments
the scIL-
12 polypeptide is administered intratumorally. In additional embodiments, the
scIL-12
polynucleotide is contained in an expression vector. In a preferred
embodiment, the
expression vector is an adenoviral vector or adeno-associated viral (AAV)
vector.
[0221] The scIL-12 polynucleotides and polypeptides of the invention may
be
administered in combination with one or more therapeutic agents and/or
procedures in the
treatment, prevention, amelioration and/or cure of cancers.

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[0222]
In a s pecific embodiment, scIL-12 polynucleotides and polypeptides of the
invention are administered in combination with one or more chemotherapeutic
useful in
the treatment of cancers including, but not limited to Alkylating agents;
Nitrogen
mustards (mechlorethamine, cyclophosphamide, ifosfamide, melphalan,
chlorambucil);
Nitrosoureas (carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU),
Ethylenimine/Methyl-melamine, thriethylenemelamine (TEM),
triethylene
thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine)); Alkyl
sulfonates (busulfan); Triazines (dacarbazine (DTIC)); Folic Acid analogs
(methotrexate,
Trimetrexate, Pemetrexed); Pyrimidine analogs (5-fluorouracil
fluorodeoxyuridine,
gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-
difluorodeoxy-
cytidine); Purine analogs (6-mercaptopurine, 6-thio guanine, azathioprine, 2'-
deoxycoformycin (pentostatin), erythrohydroxynonyl-adenine (EHNA), fludarabine
phosphate, 2-chlorodeoxyadenosine (cladribine, 2-CdA)); Type I Topoisomerase
Inhibitors (camptothecin, topotecan, irinotecan); Biological response
modifiers (IL-2, G-
CSF, GM-CSF); Differentiation Agents (retinoic acid derivatives, Hormones and
antagonists); Adrenocorticosteroids/antagonists (prednisone and equivalents,
dexamethasone, ainoglutethimide); Pro gestins (hydroxyprogesterone capro ate,
medroxyprogesterone acetate, megestrol acetate); Estrogens
(diethylstilbestrol, ethynyl
estradiol/equivalents); Antiestrogen (tamoxifen); Androgens (testosterone
propionate,
fluoxymesterone/equivalents); Antiandrogens (flutamide, gonadotropin-releasing
hormone analogs, leuprolide); Nonsteroidal antiandrogens (flutamide); Natural
products;
Antimitotic drugs; Taxanes (paclitaxel, Vinca alkaloids, vinblastine (VLB),
vincristine,
vinorelbine, Taxotere (docetaxel), estramustine, estramustine phosphate);
Epipodophylotoxins (etoposide, teniposide); Antibiotics (actimomycin D,
daunomycin
(rubido-mycin), doxorubicin (adria-mycin), mitoxantroneidarubicin, bleomycin,
splicamycin (mithramycin), mitomycinC, dactinomycin, aphidicolin); Enzymes (L-
asp araginase, L-arginase); Radio s ensitiz ers
(metronidazole, misonidazole,
desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, RSU 1069, E09,
RB
6145, SR4233, nicotinamide, 5 -bromo deozyuridine,
5 -io do deoxyuridine,
bromodeoxycytidine); Platinium coordination complexes (cisplatin, Carboplatin,
oxaliplatin, Anthracenedione, mitoxantrone); Substituted urea (hydroxyurea);
Oxazaphosphorines (cyclophosphamide; ifosfamide; trofosfamide; mafosfamide
(NSC

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345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide mustard), S-(-)-
bromofosfamide (CBM-11), NSC 612567 ( aldophosphamide perhydrothiazine); NSC
613060 (aldophosphamide thiazolidine); isophosphoramide mustard; palifosfamide
lysine); Methylhydrazine derivatives (N-methylhydrazine (MIH), procarbazine);
Adrenocortical suppressant (mitotane (o,p'-DDD), ainoglutethimide); Cytokines
(interferon (alpha, beta, gamma), interleukin-2); Photosensitizers
(hematoporphyrin
derivatives, Photofrin, benzoporphyrin derivatives, Npe6, tin etioporphyrin
(SnET2),
pheoboride-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc
phthalocyanines); and Radiation (X-ray, ultraviolet light, gamma radiation,
visible light,
infrared radiation, microwave radiation).
Modes of Administration
[0223]
The scIL-12 polypeptides and polynucleotides may be administered to the
subject
systemically or locally (e.g., at the site of the disease or disorder). S
ystemic
administration may be by any suitable method, including subcutaneously and
intravenously. Local administration may be by any suitable method, including
without
limitation, intraperitoneally, intrathecally, intraventricularly, or by direct
injection into a
tissue or organ, such as intratumoral injection.
[0224] In certain embodiments, scIL-12 polynucleotide expression is
controlled by a
ligand-inducible gene switch system, such as described, for example, in:
PCT/US2001/009050 (WO 2001/070816); U .S. Pat. Nos. 7,091,038; 7,776,587;
7,807,417; 8,202,718; P CT/US2001/030608 (WO 2002/029075); U.S. Pat. Nos.
8,105,825; 8,168,426; P CT/U52002/005235 (WO 2002/066613); U.S. App. No.
10/468,200 (U.S. Pub. No. 20120167239); P CT/U52002/005706 (WO 2002/066614);
U.S. Pat. Nos. 7,531,326; 8,236,556; 8, 598,409;
PCT/U52002/005090 (WO
2002/066612); U .S. App. No. 10/468,193 (U.S. Pub. No. 20060100416);
PCT/U52002/005234 (WO 2003/027266); U .S. Pat. Nos. 7,601,508; 7,829,676;
7,919,269; 8,030,067; P CT/U52002/005708 (WO 2002/066615); U.S. App. No.
10/468,192 (U.S. Pub. No. 20110212528); P CT/US2002/005026 (WO 2003/027289);
U.S. Pat. Nos. 7,563,879; 8,021,878; 8, 497,093;
PCT/U52005/015089 (WO
2005/108617); U .S. Pat. No. 7,935,510; 8, 076,454; P CT/U52008/011270 (WO
2009/045370); and, U.S. App. No. 12/241,018 (U.S. Pub. No. 20090136465). In
these
embodiments, once the scIL-12 polynucleotides under the control of a gene
switch have

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been introduced to the subject, an activating ligand may be administered to
induce
expression of the scIL-12 polypeptide of the invention. The ligand may be
administered
by any suitable method, either systemically (e.g., orally, intravenously) or
locally (e.g.,
intraperitoneally, intrathecally, intraventricularly, direct injection into
the tissue or organ
where the disease or disorder is occurring, including intratumorally). The
optimal timing
of ligand administration can be determined for each type of cell and disease
or disorder
using only routine techniques.
[0225] In certain embodiments, scIL-12 polynucleotides are introduced into
in vitro
engineered cells such as immune cells (e.g., dendritic cells, T cells, Natural
Killer cells)
or stem cells (e.g., mesenchymal stem cells, endometrial stem cells,
endometrial
regenerative cell (ERC), embryonic stem cells), which conditionally express a
scIL-12
polypeptide under the control of a gene switch, which can be activated by an
activating
ligand. Such methods are described in detail, for example, in: P
CT/US2008/011563
(WO 2009/048560); U.S. App. No. 12/247,738 (U.S. Pub. No. 20090123441);
PCT/U52009/005510 (WO 2010/042189); U.S. App. No. 13/123,129 (U.S. Pub. No.
20110268766); P CT/US2011/029682 (WO 2011/119773); U.S. App. No. 13/636,473
(U.S. Pub. No. 20130195800); P CT/US2012/027515 (WO 2012/122025); and, U.S.
App. No. 14/001,943 (U. S . Pub. No. [Pending]).
[0226] In one embodiment, immune cells or stem cells are transfected with
an adenovirus
vector or an adeno-associated virus vector comprising a scIL-12 polynucleotide
to
produce in vitro engineered cells.
[0227] In one embodiment the in vitro engineered immune cells or stem
cells are
autologous cells. In another embodiment the in vitro engineered immune cells
or stem
cells are allogeneic.
[0228] One embodiment of the invention provides a method for treating a
tumor,
comprising the steps in order of: 1) administering intratumorally in a mammal
a
population of in vitro engineered immune cells or stem cells containing a scIL-
12 vector
under the control of a gene switch; and 2) administering to said mammal a
therapeutically
effective amount of an activating ligand.
[0229] In certain embodiments the mammal is a human. In other embodiments
the
mammal is a dog, a cat, or a horse.

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[0230] In one embodiment, the activating ligand is administered at
substantially the same
time as the composition comprising the in vitro engineered cells or the
vector, e.g.,
adenoviral or adeno-associated viral vector, e.g., within one hour before or
after
administration of the cells or the vector compositions. In another embodiment,
the
activating ligand is administered at or less than about 24 hours after
administration of the
in vitro engineered immune cells or stem cells, or the vector. In still
another embodiment,
the activating ligand is administered at or less than about 48 hour s after
the in vitro
engineered immune cells or stem cells, or the vector. In another embodiment,
the ligand is
RG-115932. In another embodiment, the ligand is administered at a dose of
about 1 to 50
mg/kg/day. In another embodiment, the ligand is administered at a dose of
about 30
mg/kg/day. In another embodiment, the ligand is administered daily for a
period of 7 to
28 days. In another embodiment, the ligand is administered daily for a period
of 14 days.
In another embodiment, about 1x106 to 1x108 cells are administered. In another
embodiment, about 1x107 cells are administered.
[0231] Having provided for the substantially pure polypeptides,
biologically active
fragments thereof and recombinant polynucleotides encoding them, the present
invention
also provides cells comprising each of them. By appropriate introduction
techniques well
known in the field, cells comprising them may be produced. See, e.g., Sambrook
et al.
(1989).
HOST CELLS AND NON-HUMAN ORGANISMS
[0232] Another aspect of the present invention involves cells comprising
an isolated
polynucleotide encoding a scIL-12 polypeptide of the present invention. In a
specific
embodiment, the invention relates to an isolated host cell comprising a vector
comprising
a polynucleotide encoding a scIL-12 polypeptide of the present invention. The
present
invention also relates to an isolated host cell comprising an expression
vector according
to the invention. In another specific embodiment, the invention relates to an
isolated host
cell comprising a gene expression cassette comprising a polynucleotide
encoding a scIL-
12 polypeptide of the present invention. In another specific embodiment, the
invention
relates to an isolated host cell transfected with a g ene expression
modulation system
comprising a polynucleotide encoding a scIL-12 polypeptide of the present
invention. In
still another embodiment, the invention relates to a method for producing a
scIL-12
polypeptide, wherein the method comprises culturing an isolated host cell
comprising a

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polynucleotide encoding a scIL-12 polypeptide of the present invention in
culture
medium under conditions permitting expression of the polynucleotide encoding
the scIL-
12 polypeptide, and isolating the scIL-12 polypeptide from the culture.
[0233] In one embodiment, the isolated host cell is a prokaryotic host
cell or a eukaryotic
host cell. In another specific embodiment, the isolated host cell is an
invertebrate host
cell or a vertebrate host cell. Preferably, the isolated host cell is selected
from the group
consisting of a bacterial cell, a fungal cell, a yeast cell, a nematode cell,
an insect cell, a
fish cell, a plant cell, an avian cell, an animal cell, and a mammalian cell.
For example
but without limitation, the isolated host cell may be a yeast cell, a nematode
cell, an insect
cell, a plant cell, a zebrafish cell, a chicken cell, a hamster cell, a mouse
cell, a rat cell, a
rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a cow cell, a
pig cell, a horse
cell, a sheep cell, or a non-human primate cell (for example, a simian cell, a
monkey cell,
a chimpanzee cell), or a human cell.
[0234] Examples of host cells include, but are not limited to, fungal or
yeast species such
as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or
bacterial
species such as those in the genera Synechocystis, Synechococcus, Salmonella,
Bacillus,
Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas,
Methylomonas,
Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus,
Methanobacterium
and Klebsiella; animal; and mammalian host cells.
[0235] In one embodiment, the isolated host cell is a yeast cell selected
from the group
consisting of a Saccharomyces, a Pichia, and a Candida host cell.
[0236] In another embodiment, the isolated host cell is a Caenorhabdus
elegans
nematode cell.
[0237] In another embodiment, the isolated host cell is a mammalian cell
selected from
the group consisting of a hamster cell, a mouse cell, a rat cell, a rabbit
cell, a cat cell, a
dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, a horse cell, a
sheep cell, a non-
human primate cell (such as a monkey cell or a chimpanzee cell), and a human
cell.
[0238] Host cell transformation is well known in the art and may be
achieved by a variety
of methods including but not limited to electroporation, viral infection,
plasmid/vector
transfection, non-viral vector mediated transfection, Agrobacterium-mediated
transformation, particle bombardment, and the like. Expression of desired gene
products
involves culturing the transformed host cells under suitable conditions and
inducing

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expression of the transformed gene. Culture conditions and gene expression
protocols in
prokaryotic and eukaryotic cells are well known in the art (see General
Methods section
of Examples). C ells may be harvested and the gene products isolated according
to
protocols specific for the gene product.
[0239] In addition, a host cell may be chosen that modulates the
expression of the
transfected polynucleotide, or modifies and processes the polypeptide product
in a
specific fashion desired. Different host cells have characteristic and
specific mechanisms
for the translational and post-translational processing and modification
[e.g.,
glycosylation, cleavage (e.g., of signal sequence)] of proteins. Appropriate
cell lines or
host systems can be chosen to ensure the desired modification and processing
of the
foreign protein expressed. For example, expression in a bacterial system can
be used to
produce a non-glycosylated core protein product. However, a polypeptide
expressed in
bacteria may not be properly folded. E xpression in yeast can produce a
glycosylated
product. E xpression in eukaryotic cells can increase the likelihood of
"native"
glycosylation and folding of a heterologous protein. Moreover, expression in
mammalian
cells can provide a tool for reconstituting, or constituting, the
polypeptide's activity.
Furthermore, different vector/host expression systems may affect processing
reactions,
such as proteolytic cleavages, to a different extent.
[0240] Applicants' invention also relates to a non-human organism
comprising an
isolated host cell according to the invention. In a specific embodiment, the
non-human
organism is a prokaryotic organism or a eukaryotic organism. I n another
specific
embodiment, the non-human organism is an invertebrate organism or a v
ertebrate
organism.
[0241] In certain embodiments, the non-human organism is selected from the
group
consisting of a bacterium, a fungus, a yeast, a nematode, an insect, a fish, a
plant, a bird,
an animal, and a m ammal. More preferably, the non-human organism is a yeast,
a
nematode, an insect, a plant, a zebrafish, a chicken, a hamster, a mouse, a
rat, a rabbit, a
cat, a dog, a bovine, a goat, a cow, a pig, a horse, a sheep, or a non-human
primate (such
as a simian, a monkey, or a chimpanzee).
[0242] The present invention may be better understood by reference to the
following non-
limiting Examples, which are provided as exemplary of the invention.

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EXAMPLES
General molecular biology techniques
[0243] In accordance with the present invention there may be employed
conventional
molecular biology, microbiology, and recombinant DNA techniques within the
skill of
the art. Such techniques are explained fully in the literature. See, e.g.,
Green &
Sambrook, Molecular Cloning: A Laboratory Manual, Fourth Edition (2012) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Green &
Sambrook, 2012"); DNA Cloning: A Practical Approach, Volumes I and II, Second
Edition (D.M. Glover and B.D. Hames, eds. 1995); Oligonucleotide Synthesis
(M.J. Gait
ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)];
Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)];
Culture of
Animal Cells: A Manual of Basic Technique and Specialized Applications [R.I.
Freshney
(2010)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A
Practical
Guide To Molecular Cloning, Second Edition (1988); F.M. Ausubel et al. (eds.),
Current
Protocols in Molecular Biology, John Wiley & Sons, Inc. (2013).
[0244] Conventional cloning vehicles include pBR322 and pUC type plasmids
and
phages of the M13 series. These may be obtained commercially (e.g., Life
Technologies
Corporation; Promega Corporation).
[0245] For ligation, DNA fragments may be separated according to their
size by agarose
or acrylamide gel electrophoresis, extracted with phenol or with a
phenol/chloroform
mixture, precipitated with ethanol and then incubated in the presence of phage
T4 DNA
ligase (New England Biolabs, Inc.) according to the supplier's
recommendations.
[0246] The filling in of 5' protruding ends may be performed with the
Klenow fragment
of E. coli DNA polymerase I (New England Biolabs, Inc.) according to the
supplier's
specifications. The destruction of 3' protruding ends is performed in the
presence of phage
T4 DNA polymerase (New England Biolabs, Inc.) used according to the
manufacturer's
recommendations. The destruction of 5' protruding ends is performed by a
controlled
treatment with 51 nuclease.
[0247] Mutagenesis directed in vitro by synthetic oligodeoxynucleotides
may be
performed according to the method developed by Taylor et al. [Nucleic Acids
Res. 13
(1985) 8749-8764] using commercial kits such as those distributed by Life
Technologies
Corp. and Agilent Technologies, Inc.

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[0248] The enzymatic amplification of DNA fragments by PCR [Polymerase-
catalyzed
Chain Reaction, Saiki R.K. et al., Science 230 ( 1985) 1350-1354; Mullis K.B.
and
Faloona F.A., Meth. Enzym. 155 (1987) 335-350] technique may be performed
using a
"DNA thermal cycler" (Life Technologies Corp.) according to the manufacturer's
specifications.
[0249] Verification of nucleotide sequences may be performed by the method
developed
by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using
commercial
kits such as those distributed by GE Healthcare and Life Technologies Corp.
[0250] Plasmid DNAs may be purified by the Qiagen Plasmid Purification
System
according to the manufacture's instruction.
Example 1: Design of scIL-12 fusion proteins
[0251] Single chain IL-12 molecules were designed to have one of two
configurations,
illustrated in Figure 1:
1) The p40-linker-p35 configuration (Figure 1A) contains the full-length
p40 subunit
(including wild type signal peptide) fused to the mature p35 s ubunit (without
signal peptide) via a peptide linker;
2) The p35-linker-p40 configuration (Figure 1B) contains the full-length
p35 subunit
(including wild type signal peptide) fused to the mature p40 s ubunit (without
signal peptide) via a peptide linker; and
3) The p4ON-p35-p40C insert configuration (Figure 1C) comprising, from N-
to C-
terminus :
(i) a first IL-12 p40 domain (p4ON),
(ii) an optional first peptide linker,
(iii) an IL-12 p35 domain,
(iv) an optional second peptide linker, and
(v) a second IL-12 p40 domain (p40C).
[0252] Specific human scIL-12 constructs are summarized in Table 1. Amino
acid
residues specified by number in the Description column refer to the amino acid
numbering of the full-length human p40 or p35 subunits shown in SEQ ID NOs: 2
and 4,
respectively. For example, the nucleic acid and amino acid sequences of scIL-
12
Construct ID 1481273, corresponding to SEQ ID NOs: 9 and 10, respectively, is
a p4ON-
p35-p40C insert configuration; and was designed to contain, from N- to C-
terminus, a

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first p40 domain (p4ON) consisting of amino acids 1 to 293 of SEQ ID NO: 2, a
first
linker sequence of TPS (Thr-Pro-Ser; SEQ ID NO: 41), a mature p35 sequence
consisting
of amino acids 57 to 253 of SEQ ID NO: 4, a second peptide linker sequence of
GPAPTS
(Gly-Pro-Ala-Pro-Thr-Ser; SEQ ID NO: 42), and a second p40 domain (p40C)
consisting
of amino acids 294 to 328 of SEQ ID NO: 2.
[0253] Construct ID 1481272 (SEQ ID NOs: 11 and 12) is also a p4ON-p35-40C
insert
configuration, but the p35 insert occurs between amino acid residues 259 and
260 of the
p40 subunit.
[0254] The remaining scIL-12 designs (Construct IDs 1480533 to 1480546)
represent
p40-p35 or p35-p40 single chain IL-12 molecules with various linkers as
indicated in
Table 1.
[0255] Parallel mouse constructs were also designed, using the mouse p40 a
nd p35
sequences (SEQ ID NOs: 5-8) instead of human IL-12 sequences.

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Table 1: Human scIL-12 constructs
DNA Protein
Construct
SEQ SEQ Description
ID
ID NO ID NO
1481273 9 10 p40N(l -293)-TPS-p35(57- 253)-GPAPTS-p40C(294-328)
1481272 11 12 p4ON(l-259)-GS-p35(57-253)-PQTPGP-p40C(260-328)
1480533 13 14 p40(1-328)- RSPVSGDNAFPAPTG-p35(57-253)
1480534 15 16 p40(1-328)- RSQPVPTRDLEVPLTG- P35(57-253)
1480535 17 18 p40(1-328)- RSGTPPQTGLEKPTGTG- P35(57-253)
1480536 19 20 P40(1-328)- SDVTGNTGNATYTIT- p35(57-253)
1480537 21 22 p40(1-328)- GSPKDGPEIPPTGGT- P35(57-253)
1480538 23 24 p40(1-328)- GRNAPGSPPTGNYKLEP- p35(57_253)
1480539 25 26 p40(1-328)- QKGSVGFTDPEVHQSTNL- P35(57-253)
1480540 27 28 p40(1-328)- GNVPELPDTTEHSRT- P35(57-253)
1480541 29 30 p40(1-328)- GRSHPVQPYPGAFVKEPIP- P35(57_253)
1480542 31 32 P40(1-328)- PERKERISEQTYQLS- p35(57-253)
1480543 33 34 P40(1-328)-(G4S)3- P35(57-253)
1480544 35 36 P40(1-328)-G6S- P35(57-253)
1480545 37 38 p35(35-253y RSDVNSRTGPSGATPPSGNPYTITG-p40(23-328)
1480546 39 40 p35(35-253)- PAPTPSNGSPKDGPEIPPTGG- p40(23-328)
[0256] Embodiments of the invention include, without limitation, the scIL-
12 constructs
indicated in Table 1 above. The scIL-12 constructs of the invention may
comprise, or
may not comprise, a signal peptide sequence (whether synthesized with or
without a
signal peptide or as may occur as a result of polylpeptide cleavage in the
secreted form
subsequent to in vitro or in vivo expression and post-translational
processing). For
example, but without limitation, with respect to scIL-12 Construct No. 1481273
(p4ON(1_
293)-TPS-p35(57- 253)-GPAPTS-p40C(294-328)) embodiments of the invention also
include
this polypeptide sequence without a signal peptide (e.g., p4ON(23-293)-TPS-
P35(57- 253)-
GPAPTS-p40C(294-328). Likewise, without limitation, embodiments of the
invention
include any of the remaining scIL-12 constructs shown in Table 1 without a
signal
peptide.

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Example 2: Expression of scIL-12 fusion proteins in CHO cells
[0257] Vectors were constructed containing either human or murine scIL-12
(in all cases
cloned between NheI and ClaI sites) along with a 5'UTR element derived from
human
GAPDH, a synthetic 3'UTR element and with transgene expression under control
of a
constitutive CMV promoter. Vectors encoding human or mouse scIL-12 constructs
were
transiently transfected into CHO-Kl cells (ATCC Accession CCL-61) in
triplicate using
standard high-throughput transfection methods. Briefly, CHO-Kl cells were
trypsinized,
counted and re-suspended at 120,000 cells/ml in whole growth media (F12-Ham
(Sigma)
+ L-Glutamine (Gibco)+ 10% FBS (Atlanta Biologicals). One-hundred fifty (150)
micro
liters of the cell suspension was added to a 96-well cell culture plate
(Corning). Plasmid
DNA was prepared at 100 ng/[il in sterile water and complexed with Fugene 6
reagent
(Promega) at a 3:1 DNA to Fugene 6 ratio. Five (5) micro liters of the
DNA/Fugene6
complex was added to the 96-well plate containing the cells. The cells were
then
incubated at 37 C for 48 hours. Following incubation the culture supernatant
was
harvested, and frozen at -80 C until used for ELISA assays. Positive controls
included
vectors expressing two-chain IL-12 (p35-IRES-p40 and p4O-IRES-p35, labeled in
Figure
2 as bars A and D, respectively). Culture supernatants from transfected CHO-Kl
cells
were diluted 1:10, 1:100, and 1:1000 in R&D Systems Reagent Diluent + 10%
conditioned CHO-Kl media.
[0258] Expression of scIL-12 was detected by ELISA assays run according to
the
manufacturer's instructions. R&D Systems, catalog #DY419 (mouse IL-12 ELISA)
and
#DY1270 (human IL-12 ELISA). Nine samples per vector were analyzed.
[0259] Human scIL-12 expression was detected in 20 of the 36 vectors
evaluated, and
ranged from 500 pg/mL to 900 ng/mL. See Figure 2. Mouse scIL-12 expression was
detected in 18 of the 36 vectors tested. Mouse scIL-12 expression ranged from
385
pg/mL to 1.8 pg/mL (data not shown). For both human and mouse constructs, the
p40-
linker-p35 configuration demonstrated higher expression levels than the p35-
linker-p40
configuration and two-chain (bicistronic) IL-12, suggesting that scIL-12 with
p40-linker-
p35 topology has enhanced expression, folding and/or heterodimeric assembly as
compared to the p35-linker-p40 single chain configuration and two-chain IL-12.
[0260] Surprisingly, the human scIL-12 construct ID 1481273, having the
configuration:
p40N(1 to 293)- TPS-P35(57- 253)-GPAPTS-p40C(294 to 328)

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resulted in scIL-12 protein expression that was similar to levels produced by
two-chain
(bicistronic) vectors (p40-IRES-p35 and p35-IRES-p40) and single chain p35-
linker-p40
configuration, although not as high as the p40-linker-p35 configuration. S ee
Figure 2.
Similar expression patterns were observed for the mouse scIL-12 designs.
Construct ID
1481272, having the configuration p4ON(l-259)-GS-p35(57-253)-PQTPGP-p40C(26o-
328) 5 was
found not to express detectable protein.
Example 3: scIL-12 stimulation of IFN-gamma production in NK cells
[0261] Natural Killer (NK) cells secrete interferon gamma (IFN-gamma) in
response to
IL-12 exposure. Therefore, we measured IFN-gamma production in NK-92 cells
(ATCC
Accession CRL-2407), a human Natural Killer cell line, in a bioassay to detect
the
functional activity of scIL-12 designs of the invention.
[0262] NK-92 cells were cultured according to the manufacturer's
instructions using the
recommended culture medium (Alpha Minimum Essential medium without
ribonucleosides and deoxyribonucleosides, with 2 m M L-glutamine; 1.5 g/L
sodium
bicarbonate; 0.2 mM inositol; 0.1 mM 2-mercaptoethanol; 0.02 mM folic acid;
100-200
U/ml recombinant IL-2; adjusted to a final concentration of 12.5% horse serum
and
12.5% fetal bovine serum). The NK-92 cells were sub-cultured 24-48 hours prior
to use in
the assay. On the day of the assay, the NK-92 cells were counted by staining
with Trypan
Blue and seeded into 96-well plates at 5 x 104 cells per well. CHO-K1/scIL-12
culture
supernatants obtained in Example 2 were diluted 1:5 in NK-92 whole growth
media and
added to the NK-92 cells. C ontrols included culture supernatants from un-
transfected
CHO-Kl cells (labeled "Mock" in Fig. 3) and from CHO-Kl cells transfected with
plasmid not expressing IL-12 (i.e., CMV-GFP; labeled "Negative" in Fig. 3) as
negative
controls; and a positive control consisting of commercially available
recombinant human
IL-12 (R&D Systems), which was tested at 1250 ng /ml or 125 ng /ml (left and
right
positive controls bars, respectively, in Fig. 3). NK-92 cell culture
supernatants were
harvested after 48 hours, and diluted 1:10, 1:100, and 1:1000 in R&D Systems
Reagent
Diluent. T he amount of IFN-gamma in the culture medium was determined using
the
R&D Systems Human IFN-gamma Duoset ELISA kit (Catalog #DY285). Nine samples
per vector were analyzed.

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[0263] Human scIL-12 proteins stimulated human IFN-gamma production in NK-
92.
Human IFN-gamma expression ranged from 600 pg/mL to 33 ng /mL. See Figure 3.
Similar IFN-gamma levels were observed for the mouse scIL-12 constructs.
[0264] Surprisingly, scIL-12 Construct ID 1481273, which exhibited
relatively low
protein expression levels (see Example 2), demonstrated equivalent activity to
recombinant two-chain IL-12 and to p40-p35 single chain constructs in the NK-
92
bioassay, suggesting that Construct ID 1481273 may be more active on a per-
molecule
basis.
Example 4: Exemplary IL-12 Functional Assay using NK cells
[0265] The assay described herein may be used to measure the ability of IL-
12
polypeptides (e.g., recombinantly produced heterologous p35/p40 (p70)
polypeptides and
single chain IL-12 (p70) polypeptides) to induce interferon-gamma ("IFN-gamma"
or
"IFN-g") production in immune cells (such as, but not limited to, NK-92 cells)
in a dose-
dependent manner. It is understood that those skilled in the field of the
invention may
modify assays and procedures, as well as used different assays, to measure
biological
activity of IL-12.
[0266] Natural Killer (NK) cells secrete interferon gamma (IFN-gamma) in
response to
contact with (exposure to) IL-12. Accordingly, in this assay, NK-92 cells are
stimulated
with escalating doses of recombinant human and/or mouse IL-12 for 24 hour s.
Subsequently, IFN-gamma in the NK-92 supernatant is measured by ELISA. As a
result,
IFN-gamma expression decreases as IL-12 dose decreases (or conversely, up to a
certain
level of dose saturation, IFN-gamma expression increases as IL-12 doses
increase).
[0267] Figure 4 s hows a typical result obtained in a dose-response graph
(or "curve")
using human IL-12 and mouse IL-12 where dose-dependent expression of IFN-gamma
by
NK-92 cells treated with escalating doses of IL-12 for 24 hours was measured.
In this
assay, NK-92 cells were seeded at 50,000 c ells/well and treated with 0.06 ¨
1000
nanograms (ng)/mL recombinant human or mouse IL-12. NK-92 supernatants were
harvested 24 hour s later and tested by ELISA detection of human IFN-gamma. D
ata
depicted shows average IFN-gamma expression from 3 replicate samples,
calculated
based on the 1:5 sample dilution. Error bars show standard deviation. The
ELISA was
run in the presence of 20% NK-92 conditioned media to account for endogenous
IFN-
gamma expression from cells. The results demonstrate that IFN-gamma expression
from

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NK-92 cells is IL-12 dose-dependent. Notably, NK-92 cells responded similarly
to both
human and mouse IL-12.
[0268] The protocol used in this assay utilized NK-92 cells harvested and
centrifuged at
1200 rpm for 5 minutes. Spent media was removed and replaced with 1/5 volume
of
fresh medium. Cells were counted using a hemacytometer and resuspended at
lx106
cells/mL. Fifty microliters per well of NK-92 cells were plated into 96 well
tissue-culture
treated plates and incubated at 37 C with 5% CO2 incubator until ready to
dose. A
dilution curve of rhIL-12 (recombinant human IL-12) or rmIL-12 (recombinant
mouse
IL-12) was prepared by diluting IL-12 in NK-92 culture media at final
concentrations of
1000, 250, 62.5, 15.63, 3.91, 0.98, 0.24 and 0.06 nanograms/mL of IL-12. Each
well of a
96-well plate (with NK92 cells) was dosed with 50 microliters per well of IL-
12; plates
were then incubated for 24 hours.
[0269] NK92 cell plates were subsequently centrifuged and cell culture
supernatants were
harvested and stored at 4 de grees C until ready to assay (for IFN-gamma). F
or the
Interferon-gamma ELISA, a s tandard curve of recombinant protein was prepared
at
concentrations of 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 0 picograms
(pg)/mL of IFN-
gamma. ELISA analysis was performed using standard procedures; comparing IFN-
gamma standards to 1:5, 1:25, 1:125, 1:625 and 1:3125 dilutions of NK92 cell
supernatants. Results obtained are shown in Figure 4.
Example 5: Production and Biological Activity Testing of Single Chain IL-12
Constructs
[0270] Experiments were performed to express single chain IL-12 constructs
in 293T
cells and measure IL-12 dose-response biological activity (i.e., ability of IL-
12
polypeptides to stimulate IFN-gamma production from NK-92 cells in a dose-
dependent
manner). These experiments further show that single chain IL-12 (scIL-12)
polypeptides
of the invention (wherein the length of linker sequences, if any, is minimized
by inserting
IL-12 p35 pol ypeptide sequences within an IL-12 p40 pol ypeptide) retains
dose-
dependent IL-12 biological activity similar to that of native IL-12.
[0271] Three IL-12 constructs were expressed by transient transfection of
293T cells for
72 hours. Transfected supernatants were harvested and IL-12 p70 (i.e., p35/p40
heterodimers or single chain IL-12 polypeptides) was quantitated by ELISA. IL-
12
constructs were then tested in a functional assay by treating NK-92 cells with
escalating
doses of IL-12 (0.00001 ¨ 100 nanograms/mL). Recombinant human IL-12
(previously

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demonstrated to induce dose-dependent expression of IFN-gamma from NK-92
cells) was
included as a positive control. 293T cell supernatants from cells transfected
with a GFP
control vector was included as a negative control. Results show that both
single chain
and native IL-12 proteins induced dose-dependent IFN-gamma expression by NK-92
cells. Furthermore, the level of induction was similar across each of the
three IL-12
constructs as well as the positive control. No IFN-gamma expression was
observed from
NK-92 cells treated with 293T GFP-transfected supernatants. See, Figure 5.
Table 2: IL-12 Constructs For Induction of Interferon-Gamma by NK92 Cells
IL-12 Construct IL-12 Linker DNA Vector
(SEQ ID Nos)
p40-linker-p35 -GGGGGGS- 275562
(SEQ ID NOs: 35 & 36)
p4ON-p35-p40C -TPS-p35-GPAPTS- 275566
(SEQ ID NOs: 9 & 10)
None
p40/p35 (Heterodimeric p35/p40 wit 275567
(SEQ ID NOs: 1/3 & 2/4) IRES separating p35 and
p40 open reading frames)
CMV-GFP N/A 40022
IFN-gamma Quantitation Procedure
[0272] Cell Seeding: One day prior to transfection, 293T cells were seeded
in a 6 well
dish at 7.58e5 cells/well (media composition of 10% FBS, DMEM, lx GLUTAMAXTm
(Life Technologies Inc.)) and incubated overnight at 37 de grees C in air
supplemented
with 5% carbon dioxide.
[0273] Transfection: Next day, DNA vectors were diluted to a final
concentration of 100
micrograms/mL DNA (starting DNA concentrations ranged from 1000 t o ¨1300
micrograms/mL) . Transfection mixes were prepared with 22 microliters FUGENEO
6
transfection reagent (Promega Corp., Madison, WI, USA), 308 microliters OPTI-
MEMO
cell culture media (Life Technologies Inc., Grand Island, NY, USA), and 36.7
microliters
DNA solution in a 15 mL conical polystyrene tube. Tube was agitated quickly
but gently

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to mix and incubated at 15 minutes at room temperature. 167 microliters of
transfection
mixture was added to each well in 6-well dishes with vectors 275566 and 275567
in
duplicate wells. Plates were incubated at 37 degrees C with 5% carbon dioxide.
Seventy-
two hours post transfection, cell culture supernatants were harvested and
sterile filtered
using a 0.2 micron filter and syringe. One-hundred and fifty microliters per
sample was
used for IL-12 ELISA quantitation. The remainder was stored at -80 degrees C
until used
in IFN-gamma assay.
[0274] IL-12 ELISA: Commercially available IL-12 ELISA kits (e.g., Human
IL-12 p40
(and p70) DUOSETO ELISA from R&D Systems Inc., Minneapolis, MN, USA) were
used according to manufacturer's directions for quantitation of IL-12 in cell
culture
supernatants. 0 ptical absorbance of ELISA plates at 450 nm were measured. C
ell
culture supernatants were determined to have the following concentrations of
IL-12:
p4ON-p35-p40C (vector 275566) at 8364 ng/mL; p40/p35 heterodimer (vector
275567) at
28903 ng/mL; and, p40-linker-p35 (vector 275562) at 57197 ng/mL. IL-12 cell
culture
supernatants were diluted to a final concentration of 2000 ng/mL IL-12. (293T
cell GFP-
transfected supernatants were diluted with same dilution factor as p4ON-p35-
p40C
supernatants).
[0275] NK-92 Functional Assay: On day 1, NK-92 cells were harvested and
centrifuged
at 1200 rpm for 5 minutes. Spent media was removed and replaced with 1/5
volume of
fresh medium. Cells were counted using a hemacytometer. An 88% viable cell
count at
2.03e6 c/mL was observed. Eight mL of NK-92 cells at 1e6 cells/mL was prepared
(using 4.5 mL cells plus 3.5 mL media to generate 8 mL at 1e6 c/mL). NK92
cells were
seeded at 50 microliters per well into two 96 well tissue-culture treated
plates and
incubated at 37 C/5% CO2 incubator until ready to dose with IL-12. Ten-fold
dilutions
of IL-12 were prepared to final concentrations of 200, 20, 2, 0.2, 0.02,
0.002, 0.0002 and
0.00002 ng/mL. N K-92 cells in 96-well plates were then dosed (in triplicate
or
quadruplicate at each concentration) with 50 m icroliters of IL-12 and
returned to
incubator for 24 hours. On day 2, the contents of each well in the 96-well
plates was
transferred to 96-well V-bottom plates and centrifuged at 1200 rpm for 10 m
mutes.
Supernatants were collected (cells were discarded) and stored at 4 degrees C
until used to
assay for IFN-gamma quantities. ELISA analysis was performed to quantitate IFN-
gamma production according to manufacturer's instructions using a commercially

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available kit (Human IFN-gamma DUOSETO ELISA from R&D Systems, Inc.)
compared to a standard curve of recombinant IFN-gamma. Optical absorbance at
450 nm
was measured.
[0276] Results: Figure 5 shows expression of IFN-gamma from NK-92 cells
treated with
increasing doses of IL-12 (24 hours exposure to IL-12). NK-92 cells were
seeded at
50,000 cells/well and treated with 0.00001 - 100 ng/mL recombinant human IL-
12, IL-12
expressed in 293T cells, or 293T supernatant from GFP-transfected cells
(negative
control). IL-12 induced NK-92 cell supernatants were harvested 24 hours later
and tested
by human IFN-gamma ELISA. Data show average IFN-gamma expression from 3-4
replicate samples, tested in duplicate at a 1:5 and 1:25 sample dilution
(n=12). Error bars
show standard deviation. ELISA was run in the presence of 20% NK-92
conditioned
media in order to account for endogenous IFN-gamma expression from cells. Data
demonstrates that IFN-gamma expression from NK-92 cells is IL-12 dose-
dependent for
both the transfected samples as well as the recombinant IL-12. IFN-gamma
expression
appears to be IL-12 specific, as indicated by the lack of IFN-gamma expression
from cells
treated with the GFP supernatants.